Polycyclic lpa1 antagonist and uses thereof

ABSTRACT

Described herein is the LPA1 antagonist 1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylic acid (Compound 1), or pharmaceutically acceptable salts thereof. Also described are methods of preparing the LPA1 antagonist, or pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions suitable for administration to a mammal that include the LPA1 antagonist, or pharmaceutically acceptable salt thereof, and methods of using such pharmaceutical compositions for treating LPA-dependent or LPA-mediated diseases or conditions.

RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.61/420,599, entitled “POLYCYCLIC LPA₁ ANTAGONIST AND USES THEREOF” filedon Dec. 7, 2010, which is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

Described herein is the LPA receptor antagonist1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1), pharmaceutically acceptable salts, polymorphs,amorphous phases, metabolites thereof, as well as pharmaceuticalcompositions thereof, and methods of use thereof in the treatment orprevention or diagnosis of diseases or conditions associated with theactivity of one or more of the lysophosphatidic acid (LPA) receptors.

BACKGROUND OF THE INVENTION

Lysophospholipids are membrane-derived bioactive lipid mediators.Lysophospholipids affect fundamental cellular functions that includeproliferation, differentiation, survival, migration, adhesion, invasion,and morphogensis. These functions influence many biological processesthat include, but are not limited to, neurogensis, angiogenesis, woundhealing, fibrosis, immunity, and carcinogenesis.

Lysophosphatidic acid (LPA) is a lysophospholipid that has been shown toact through sets of specific G protein-coupled receptors (GPCRs) in anautocrine and paracrine fashion. LPA binding to its cognate GPCRs (LPA₁,LPA₂, LPA₃, LPA₄, LPA₅, LPA₆) activates intracellular signaling pathwaysto produce a variety of biological responses. Antagonists of the LPAreceptors find use in the treatment of diseases, disorders or conditionsin which LPA plays a role.

SUMMARY OF THE INVENTION

Described herein is1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1), including all pharmaceutically acceptable solvates(including hydrates), prodrugs, polymorphs, amorphous phases andmetabolites thereof or a pharmaceutically acceptable salt of Compound 1including (including hydrates), prodrugs, polymorphs, amorphous phasesand metabolites thereof, and methods of uses thereof. Compound 1, aswell as the pharmaceutically acceptable salts thereof, are used in themanufacture of medicaments for the treatment or prevention of LPAmediated and/or LPA dependent diseases, disorders, or conditions.Compound 1 is a LPA₁ antagonist.

Described herein are pharmaceutical compositions comprising Compound 1,or a pharmaceutically acceptable salt thereof (e.g. the sodium salt) asthe active ingredient in the pharmaceutical composition. In someembodiments, described herein are pharmaceutical compositions comprisingcrystalline Compound 1, or solvate thereof. In some embodiments,described herein are pharmaceutical compositions comprising crystallineCompound 2, or solvate thereof. In some embodiments, described hereinare pharmaceutical compositions comprising a hydrate of crystallineCompound 2.

In one aspect, described is1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid,1-{4′-[3-methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or1-{4′-[3-methyl-4-(1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or a pharmaceutically acceptable salt, or solvate thereof. Inanother aspect, described is a pharmaceutically acceptable salt of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid,1-{4′-[3-methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or1-{4′-[3-methyl-4-(1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or solvate thereof. In some embodiments, the pharmaceuticallyacceptable salt of Compound 1 is substantially free of the S-isomer. Insome embodiments, the pharmaceutically acceptable salt of Compound 1 iscrystalline. In some embodiments, the pharmaceutically acceptable saltof Compound 1 is crystalline and is substantially free of the amorphouspharmaceutically acceptable salt.

In one aspect, described is a pharmaceutically acceptable salt of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1), or solvate thereof. In some embodiments, thepharmaceutically acceptable salt is a sodium salt, calcium salt,potassium salt, ammonium salt, L-arginine salt, L-lysine salt, orN-methyl-D-glucamine salt, or solvate thereof. In some embodiments, thepharmaceutically acceptable salt is a sodium salt, or solvate thereof.In some embodiments, the pharmaceutically acceptable salt is1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt (Compound 2), or solvate thereof. In some embodiments,the pharmaceutically acceptable salt is amorphous. In some embodiments,the pharmaceutically acceptable salt is crystalline. In someembodiments, the pharmaceutically acceptable salt is a crystalline formof Compound 2, or solvate thereof. In some embodiments, thepharmaceutically acceptable salt is a hydrated crystalline form ofCompound 2. In some embodiments, the pharmaceutically acceptable salt isan amorphous phase of Compound 2, or solvate thereof.

In one aspect, described is a crystalline form of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-S-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid,1-{4′-[3-methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or1-{4′-[3-methyl-4-(1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or a pharmaceutically acceptable salt, or solvate thereof. In someembodiments, the pharmaceutically acceptable salt is a sodium salt,calcium salt, potassium salt, ammonium salt, L-arginine salt, L-lysinesalt, or N-methyl-D-glucamine salt, or solvate thereof. In someembodiments, the pharmaceutically acceptable salt is a sodium salt, orsolvate thereof.

In some embodiments, described is a crystalline form of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl})-cyclopropanecarboxylicacid, or a pharmaceutically acceptable salt, or solvate thereof.

In some embodiments, described is a crystalline form of apharmaceutically acceptable salt of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or solvate thereof.

In any of the embodiments described herein the crystalline form ishydrated. In any of the embodiments described herein the crystallineform is a monohydrate.

In some embodiments, described is a crystalline form of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt.

In some embodiments, described is a crystalline form of a hydrate of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt.

In some embodiments, the crystalline form of the hydrate of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt (Compound 2):

-   -   (a) has an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 13.2° 2-Theta, 17.2° 2-Theta, 19.3°        2-Theta, 22.4° 2-Theta, and 25.6° 2-Theta;    -   (b) has an X-ray powder diffraction (XRPD) pattern substantially        the same as shown in FIG. 4;    -   (c) has a thermo-gravimetric analysis (TGA) or a DSC        substantially similar to the ones set forth in FIG. 5 and FIG.        6;    -   (d) has an infrared spectrum substantially similar to the one        set forth in FIG. 7:    -   (e) was obtained from methyl ethyl ketone, acetonitrile,        1,4-dioxane/tert-butyl methyl ether, methyl ethyl ketone        (MEK)/tert-butyl methyl, or ethanol/heptane;    -   (f) has unit cell parameters substantially equal to the        following at 25° C.:

a (Å) 13.8714(2) b (Å) 7.7379(2) c (Å) 25.5253(5) α° 90 β° 103.863(1) γ°90 V (Å3) 2659.96(9) Z 4 Calculated Density 1.305 Crystal SystemMonoclinic SG P2₁ R1 0.0301 Sol. Sites 1H₂O

-   -   or    -   (g) combinations thereof.

In some embodiments, the hydrated crystalline form of Compound 2 has atleast one of the properties selected from (a), (b), (c), (d), (e), and(f). In some embodiments, the hydrated crystalline form of Compound 2has at least two of the properties selected from (a), (b), (c), (d),(e), and (f). In some embodiments, the hydrated crystalline form ofCompound 2 has at least three of the properties selected from (a), (b),(c), (d), (e), and (f). In some embodiments, the hydrated crystallineform of Compound 2 has at least four of the properties selected from(a), (b), (c), (d), (e), and (f). In some embodiments, the hydratedcrystalline form of Compound 2 has at least five of the propertiesselected from (a), (b), (c), (d), (e), and (f). In some embodiments, thehydrated crystalline form of Compound 2 has properties (a), (b), (c),(d), (e), and (f).

In one embodiment, described is a crystalline form of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt (Compound 2), or a solvate thereof.

In one embodiment, described is a crystalline form of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt (Compound 2):

In some embodiments, the crystalline form of Compound 2 is hydrated.

In some embodiments, the crystalline form of Compound 2 has an X-raypowder diffraction (XRPD) pattern with characteristic peaks at 13.2°2-Theta, 17.2° 2-Theta, 19.3° 2-Theta, 22.4° 2-Theta, and 25.6° 2-Theta.

In some embodiments, the crystalline form of Compound 2 has an X-raypowder diffraction (XRPD) pattern substantially the same as shown inFIG. 4.

In some embodiments, the crystalline form of Compound 2 has a DSCthermogram substantially similar to the one set forth in FIG. 6. In someembodiments, the crystalline form of Compound 2 has a thermo-gravimetricanalysis (TGA) substantially similar to the one set forth in FIG. 5.

In some embodiments, the crystalline form of Compound 2 has athermo-gravimetric analysis (TGA) or a DSC substantially similar to theones set forth in FIG. 5 and FIG. 6.

In some embodiments, the crystalline form of Compound 2 was obtainedfrom:

-   -   (i) methyl ethyl ketone;    -   (ii) methyl ethyl ketone, methyl tert-butyl ether and water;    -   (iii) methyl ethyl ketone, and water;    -   (iv) acetonitrile or acetonitrile and tetrahydrofuran;    -   (v) 1,4-dioxane and tert-butyl methyl ether;    -   (vi) methyl ethyl ketone and tert-butyl methyl; or    -   (vii) ethanol and heptane.

In some embodiments, the crystalline form of Compound 2 is substantiallyfree of the S-isomer.

In some embodiments, the crystalline form of Compound 2 is substantiallyfree of the amorphous phase of Compound 2.

In some embodiments, the crystalline form of Compound 2 hassubstantially the same X-ray powder diffraction (XRPD) pattern after aweek's storage at elevated relative humidity.

In some embodiments, the crystalline form of Compound 2 hassubstantially the same X-ray powder diffraction (XRPD) pattern after aweek's storage at 40° C./75% relative humidity or 25° C./95% relativehumidity.

In one aspect, described herein is amorphous1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt (Compound 2). In some embodiments, amorphous Compound2 is substantially free of the S-isomer.

In some embodiments, the crystalline form of Compound 2 is Pattern 1. Insome embodiments, the crystalline form of Compound 2 is Pattern 2. Insome embodiments, the crystalline form of Compound 2 is Pattern 3.

In some embodiments, described is a crystalline form of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1), or solvate thereof.

In some embodiments, described is a crystalline form of a compound withthe following structure:

In some embodiments, the crystalline form of Compound 1 is characterizedas having:

-   -   (a) an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 4.7° 2-Theta, 9.4° 2-Theta, 14.5°        2-Theta, and 21.0° 2-Theta;    -   (b) an X-ray powder diffraction (XRPD) pattern substantially the        same as the XRPD shown in FIG. 1;    -   (c) a DSC thermogram with an endotherm at about 172° C.-176° C.;    -   (d) a DSC or a thermo-gravimetric analysis (TGA) substantially        similar to the ones set forth in FIG. 2 and FIG. 3;    -   (e) substantially the same X-ray powder diffraction (XRPD)        pattern post storage at 40 C/75% relative humidity for one week;    -   (f) unit cell parameters substantially equal to the following at        25° C.:

a (Å) 26.2070(8) b (Å) 37.700(1) c (Å) 5.0051(2) α° 90 β° 90 γ° 90 V(Å3) 4945.1(3) Z 8 Calculated Density 1.296 Crystal System OrthorhombicSG P2₁2₁2 R1 0.0418 Sol. Sites —

-   -   or    -   (g) combinations thereof.

In some embodiments, the crystalline form of Compound 1 has an X-raypowder diffraction (XRPD) pattern with characteristic peaks at 4.7°2-Theta, 9.4° 2-Theta, 14.5° 2-Theta, and 21.0° 2-Theta.

In some embodiments, the crystalline form of Compound 1 has an X-raypowder diffraction (XRPD) pattern substantially the same as the XRPDshown in FIG. 1.

In some embodiments, the crystalline form of Compound 1 has a DSCthermogram with an endotherm at about 176° C.

In some embodiments, the crystalline form of Compound 1 has a DSC or athermo-gravimetric analysis (TGA) substantially similar to the ones setforth in FIG. 2 and FIG. 3.

In some embodiments, the crystalline form of Compound 1 hassubstantially the same X-ray powder diffraction (XRPD) pattern poststorage at 40 C/75% relative humidity for one week.

In some embodiments, the crystalline form of Compound 1 is characterizedas having:

-   -   (a) an X-ray powder diffraction (XRPD) pattern substantially the        same as the XRPD shown in FIG. 12;    -   (b) an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 6.3° 2-Theta, 12.8° 2-Theta, 16.4°        2-Theta, 17.0° 2-Theta, and 19.7° 2-Theta;    -   (c) unit cell parameters approximately equal to the following at        a temperature of 25° C.:

a (Å) 30.3522(9) b (Å) 7.8514(3) c (Å) 22.4570(7) α° 90 β° 111.665(2) γ°90 V (Å³) 4973.6(3) Z 8 Calculated Density 1.289 Crystal SystemMonoclinic SG C2 R1 0.0298 Sol. Sites —

-   -   or    -   (d) combinations thereof.

In some embodiments, the crystalline form of Compound 1 is characterizedas having:

-   -   (a) an X-ray powder diffraction (XRPD) pattern substantially the        same as the XRPD shown in FIG. 13;    -   (b) an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 5.5° 2-Theta, 5.9° 2-Theta, 12.6°        2-Theta, and 16.7° 2-Theta;    -   (c) unit cell parameters approximately equal to the following at        a temperature of 25° C.:

a (Å) 32.3574(9) b (Å) 5.1057(2) c (Å) 33.148(1) α° 90 β° 114.846(2) γ°90 V (Å³) 4969.4(3) Z 8 Calculated Density 1.290 Crystal SystemMonoclinic SG C2 R1 0.0553 Sol. Sites —

-   -   or    -   (d) combinations thereof.

In some embodiments, the crystalline form of Compound 1 is substantiallyfree of the S-isomer.

In some embodiments, the crystalline form of Compound 1 is substantiallyfree of amorphous Compound 1.

In some embodiments, the crystalline form of Compound 1 is crystallizedfrom ethanol, methanol, 2-methoxyethanol, ethanol, 1-propanol,2-propanol, 1-butanol, butyl acetate, acetone, methylethyl ketone,anisole, toluene, nitromethane, acetonitrile, ethyl acetate, cumene,1-4-dioxane, tetrahydrofuran, dichloromethane, heptane, or combinationsthereof.

In some embodiments, the crystalline form of Compound 1 is Pattern 1. Insome embodiments, the crystalline form of Compound 1 is Pattern 2. Insome embodiments, the crystalline form of Compound 1 is Pattern 3.

In some embodiments, the pharmaceutically acceptable salt of Compound 1includes a detectable amount of palladium that is less than 20 ppm. Insome embodiments, the pharmaceutically acceptable salt of Compound 1includes a detectable amount of palladium that is less than 15 ppm. Insome embodiments, the pharmaceutically acceptable salt of Compound 1does not include a detectable amount of palladium.

In one aspect, provided is a compound which has the following structure:

or a pharmaceutically acceptable salt thereof.

In some embodiments, described herein is a pharmaceutical compositioncomprising Compound 1, or a pharmaceutically acceptable salt or solvatethereof. In some embodiments, Compound 1, or the pharmaceuticallyacceptable salt or solvate thereof is amorphous. In some embodiments,Compound 1, or the pharmaceutically acceptable salt or solvate thereofis crystalline.

In some embodiments, described herein are pharmaceutical compositionscomprising a crystalline form of Compound 1 or a pharmaceuticallyacceptable salt or solvate thereof. In some embodiments, describedherein are pharmaceutical compositions comprising a crystalline form ofCompound 2, or solvate thereof. In some embodiments, described hereinare pharmaceutical compositions comprising a hydrated crystalline formof Compound 2. In some embodiments, described herein are pharmaceuticalcompositions comprising Compound 2 (Pattern 1).

In some embodiments, the pharmaceutical composition comprises at leastinactive ingredient selected from pharmaceutically acceptable carriers,diluents and excipients.

In some embodiments, the pharmaceutical composition comprises Compound2, or a solvate thereof.

In some embodiments, the pharmaceutical composition comprisescrystalline Compound 2, or solvate thereof.

In some embodiments, Compound 2, or solvate thereof is greater than 96%pure. In some embodiments, Compound 2, or solvate thereof is greaterthan 97% pure. In some embodiments, Compound 2, or solvate thereof isgreater than 98% pure.

In some embodiments, the pharmaceutical composition is formulated forintravenous injection, subcutaneous injection, oral administration,inhalation, nasal administration, topical administration, ophthalmicadministration or otic administration.

In some embodiments, the pharmaceutical composition is a tablet, a pill,a capsule, a liquid, an inhalant, a nasal spray solution, a suppository,a suspension, a gel, a colloid, a dispersion, a suspension, a solution,an emulsion, an ointment, a lotion, an eye drop or an ear drop.

In some embodiments, the pharmaceutical composition is in a formsuitable for oral administration to a mammal.

In some embodiments, the pharmaceutical composition is in the form of apill, capsule, tablet, aqueous solution, aqueous suspension, non-aqueoussolution, or non-aqueous suspension.

In some embodiments, the pharmaceutical composition is in the form of acapsule. In some embodiments, the pharmaceutical composition is in theform of an immediate release capsule or an enteric coated capsule. Insome embodiments, the capsule is a hard gelatine capsule or hypromellose(HPMC) capsule. In some embodiments, the capsule comprises at least oneexcipient in addition to the hard gelatine capsule or hypromellose(HPMC) capsule.

In some embodiments, the pharmaceutical composition is in the form of atablet. In some embodiments, the pharmaceutical composition is in theform of an immediate release tablet, an enteric coated tablet, or asustained release tablet. In some embodiments, the pharmaceuticalcomposition is in the form of a moisture barrier coated tablet.

In some embodiments, the pharmaceutical composition is in the form of anaqueous solution or aqueous suspension.

In some embodiments, a single dose of the pharmaceutical compositioncomprises about 10 mg to about 1500 mg of Compound 1, or apharmaceutically acceptable salt or solvate thereof.

In some embodiments, a single dose of the pharmaceutical compositioncomprises about 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, about 100 mg, about150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about800 mg, about 900 mg or about 1000 mg of Compound 1, or apharmaceutically acceptable salt or solvate thereof (e.g. Compound 2).

In some embodiments, a single dose of the pharmaceutical compositioncomprises about 10 mg to about 1500 mg of Compound 2, or solvatethereof.

In some embodiments, a single dose of the pharmaceutical compositioncomprises about 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, about 100 mg, about150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about800 mg, about 900 mg or about 1000 mg of Compound 2, or solvate thereof.

In some embodiments, the pharmaceutical compositions described hereincomprise a detectable amount of a compound with the structure:

In some embodiments, described herein is a pharmaceutical compositionthat provides at least one metabolite of Compound 1 after administrationto a mammal.

In some embodiments, the at least one metabolite is selected from among:

-   -   glucuronidation of Compound 1;    -   glucuronidation of Compound 1 plus oxidation;

In some embodiments, described herein is a method of inhibiting thephysiological activity of LPA in a mammal comprising administeringCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2), or a pharmaceutical composition thereof to the mammal in needthereof.

In some embodiments, described herein is a method for treating orpreventing a LPA-dependent or LPA-mediated disease or condition in amammal comprising administering Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2), or a pharmaceuticalcomposition thereof to the mammal in need thereof.

In some embodiments, the LPA-dependent or LPA-mediated disease orcondition is selected from lung fibrosis, asthma, chronic obstructivepulmonary disease (COPD), renal fibrosis, acute kidney injury, chronickidney disease, liver fibrosis, skin fibrosis, fibrosis of the gut,breast cancer, pancreatic cancer, ovarian cancer, prostate cancer,glioblastoma, bone cancer, colon cancer, bowel cancer, head and neckcancer, melanoma, multiple myeloma, chronic lymphocytic leukemia, cancerpain, tumor metastasis, transplant organ rejection, scleroderma, ocularfibrosis, age related macular degeneration (AMD), diabetic retinopathy,collagen vascular disease, atherosclerosis, Raynaud's phenomenom, orneuropathic pain.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is used in the treatment or prevention offibrosis, inflammation or cancer in a mammal.

In some embodiments, described herein is a method of controlling theactivation of LPA receptors in a tissue in a mammal comprisingadministering Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), or a pharmaceutical composition thereof to the mammalin need thereof. In some embodiments, the activation of LPA receptors ina tissue in a mammal results in fibrosis.

In some embodiments, described herein is a method for the treatment orprevention of fibrosis in a mammal comprising administering Compound 1,or a pharmaceutically acceptable salt thereof (e.g. Compound 2), or apharmaceutical composition thereof to the mammal in need thereof. Insome embodiments, the fibrosis comprises lung fibrosis, renal fibrosis,hepatic fibrosis or cutaneous fibrosis.

In some embodiments, described herein is a method of improving lungfunction in a mammal comprising administering Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), or apharmaceutical composition thereof to the mammal in need thereof. Insome embodiments, the mammal has been diagnosed as having lung fibrosis.

In some embodiments, described herein is a method of treating idopathicpulmonary fibrosis in a mammal comprising administering Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), or apharmaceutical composition thereof to the mammal in need thereof.

In some embodiments, described herein is a method of controlling anabnormal accumulation or activation of cells, fibronectin, collagen orincreased fibroblast recruitment in a tissue of a mammal comprisingadministering Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), or a pharmaceutical composition thereof to the mammalin need thereof.

In some embodiments, the abnormal accumulation or activation of cells,fibronectin, collagen or increased fibroblast recruitment in the tissueresults in fibrosis.

In some embodiments, described herein is a method for the treatment orprevention of scleroderma in a mammal comprising administering Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2), or apharmaceutical composition thereof to the mammal in need thereof.

In some embodiments, described herein is a method for reducing undesiredor abnormal dermal thickening in a mammal comprising administering tomammal in need thereof Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), or a pharmaceutical composition thereof. Insome embodiments, the dermal thickening is associated with scleroderma.

In some embodiments, described herein is a method of controlling anabnormal accumulation or activation of cells, fibronectin, collagen orincreased fibroblast recruitment in dermal tissues of a mammalcomprising administering to mammal in need thereof Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), or apharmaceutical composition thereof. In some embodiments, the abnormalaccumulation or activation of cells, fibronectin, collagen or increasedfibroblast recruitment in the dermal tissues results in dermal fibrosis.In some embodiments, described herein is a method of reducinghydroxyproline content in dermal tissues of a mammal with cutaneousfibrosis comprising administering to mammal in need thereof Compound 1,or a pharmaceutically acceptable salt thereof (e.g. Compound 2), or apharmaceutical composition thereof.

In some embodiments, described herein is a method for the treatment orprevention of Raynaud's phenomenom in a mammal comprising administeringCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2), or a pharmaceutical composition thereof to the mammal in needthereof.

In some embodiments, the pharmaceutical composition is administereddaily to the mammal. In some embodiments, the pharmaceutical compositionis administered once-daily to the mammal. In some embodiments, thepharmaceutical composition is administered twice-daily to the mammal.

In some embodiments, the mammal is a human.

In some embodiments, in any of the method of treatments involving amammal, the mammal is administered one or more additionaltherapeutically active agents in addition to Compound 1, or apharmaceutically acceptable salt thereof.

In some embodiments, in any of the method of treatments involving amammal, the mammal is administered one or more additionaltherapeutically active agents selected from: corticosteroids,immunosuppresant, analgesics, anti-cancer agent, anti-inflammatories,chemokine receptor antagonists, bronchodilators, leukotriene receptorantagonists, leukotriene formation inhibitors, monoacylglycerol kinaseinhibitors, phospholipase A₁ inhibitors, phospholipase A₂ inhibitors,and lysophospholipase D (lysoPLD) inhibitors, autotaxin inhibitors,decongestants, antihistamines, mucolytics, anticholinergics,antitussives, expectorants, and β-2 agonists.

In some embodiments, provided is a method comprising administeringCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2), to a human with a LPA-dependent or LPA-mediated disease orcondition. In some embodiments, the human is already being administeredone or more additional therapeutically active agents other than Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2). Insome embodiments, the method further comprises administering one or moreadditional therapeutically active agents other than Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2).

In some embodiments, the one or more additional therapeutically activeagents other than Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), are selected from: corticosteroids,immunosuppressants, analgesics, anti-cancer agent, anti-inflammatories,chemokine receptor antagonists, bronchodilators, leukotriene receptorantagonists, leukotriene formation inhibitors, monoacylglycerol kinaseinhibitors, phospholipase A₁ inhibitors, phospholipase A₂ inhibitors,and lysophospholipase D (lysoPLD) inhibitors, autotaxin inhibitors,decongestants, antihistamines, mucolytics, anticholinergics,antitussives, expectorants, and β-2 agonists.

In another aspect is the use of Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2), in the treatment of adisease, disorder or condition in which the activity of at least one LPAreceptor contributes to the pathology and/or symptoms of the disease orcondition. In one embodiment of this aspect, the LPA receptor isselected from LPA₁, LPA₂, LPA₃, LPA₄, LPA₅ and LPA₆. In someembodiments, the LPA receptor is LPA₁ or LPA₂ or LPA₃. In someembodiments, the disease or condition is any of the diseases orconditions specified herein.

Also provided is a method of inhibiting the physiological activity ofLPA in a mammal comprising administering a therapeutically effectiveamount of a compound of Compound 1, or a pharmaceutically acceptablesalt thereof (e.g. Compound 2), to the mammal in need thereof.

In one aspect, is a method for treating or preventing a LPA-dependent orLPA-mediated disease or condition in a mammal comprising administering atherapeutically effective amount of Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2).

In one aspect, LPA-dependent or LPA-mediated diseases or conditionsinclude, but are not limited to, fibrosis of organs or tissues,scarring, liver diseases, dermatological conditions, cancer,cardiovascular disease, respiratory diseases or conditions, inflammatorydisease, gastrointestinal tract disease, renal disease, urinarytract-associated disease, inflammatory disease of lower urinary tract,dysuria, frequent urination, pancreas disease, arterial obstruction,cerebral infarction, cerebral hemorrhage, pain, peripheral neuropathy,and fibromyalgia.

In some embodiments, the LPA-dependent or LPA-mediated disease orcondition is selected from idiopathic pulmonary fibrosis; other diffuseparenchymal lung diseases of different etiologies including iatrogenicdrug-induced fibrosis, occupational and/or environmental inducedfibrosis, granulomatous diseases (sarcoidosis, hypersensitivitypneumonia), collagen vascular disease, alveolar proteinosis, langerhanscell granulomatosis, lymphangioleiomyomatosis, inherited diseases(Hermansky-Pudlak Syndrome, tuberous sclerosis, neurofibromatosis,metabolic storage disorders, familial interstitial lung disease);radiation induced fibrosis: chronic obstructive pulmonary disease(COPD); scleroderma: bleomycin induced pulmonary fibrosis; chronicasthma; silicosis; asbestos induced pulmonary fibrosis; acuterespiratory distress syndrome (ARDS); kidney fibrosis;tubulointerstitium fibrosis; glomerular nephritis; focal segmentalglomerular sclerosis; IgA nephropathy; hypertension; Alport; gutfibrosis; liver fibrosis; cirrhosis; alcohol induced liver fibrosis;toxic/drug induced liver fibrosis; hemochromatosis: nonalcoholicsteatohepatitis (NASH); biliary duct injury; primary biliary cirrhosis;infection induced liver fibrosis; viral induced liver fibrosis; andautoimmune hepatitis; corneal scarring; hypertrophic scarring; Duputrendisease, keloids, cutaneous fibrosis; cutaneous scleroderma; spinal cordinjury/fibrosis; myelofibrosis; vascular restenosis; atherosclerosis;arteriosclerosis; Wegener's granulomatosis; Peyronie's disease, chroniclymphocytic leukemia, tumor metastasis, transplant organ rejection,endometreosis, neonatal respiratory distress syndrome and neuropathicpain.

In one aspect, is a method for treating or preventing cancer in a mammalcomprising administering a therapeutically effective amount of Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2), tothe mammal in need thereof.

In one aspect, is a method for treating or preventing fibrosis in amammal comprising administering a therapeutically effective amount ofCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2), to the mammal in need thereof.

In one aspect, is a method for treating or preventing lung fibrosis,asthma, chronic obstructive pulmonary disease (COPD), renal fibrosis,acute kidney injury, chronic kidney disease, liver fibrosis, skinfibrosis, fibrosis of the gut, breast cancer, pancreatic cancer, ovariancancer, prostate cancer, glioblastoma, bone cancer, colon cancer, bowelcancer, head and neck cancer, melanoma, multiple myeloma, chroniclymphocytic leukemia, cancer pain, tumor metastasis, transplant organrejection, scleroderma, ocular fibrosis, age related maculardegeneration (AMD), diabetic retinopathy, collagen vascular disease,atherosclerosis, Raynaud's phenomenom, or neuropathic pain in a mammalcomprising administering a therapeutically effective amount of Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2), tothe mammal in need thereof.

In one aspect, provided is a method for the treatment or prevention oforgan fibrosis in a mammal comprising administering a therapeuticallyeffective amount of Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), to a mammal in need thereof. In someembodiments, the organ fibrosis comprises lung fibrosis, renal fibrosis,or hepatic fibrosis.

In one aspect, provided is a method of improving lung function in amammal comprising administering a therapeutically effective amount ofCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2), to the mammal in need thereof. In one aspect, the mammal has beendiagnosed as having lung fibrosis.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to treat idiopathic pulmonary fibrosis (usualinterstitial pneumonia) in a mammal.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to treat Raynaud's phenomenon. Raynaud'sphenomenon comprises both Raynaud's disease (where the phenomenon isidiopathic) and Raynaud's syndrome, where it is caused by some otherinstigating factor.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is used to treat diffuse parenchymalinterstitial lung diseases in mammal: iatrogenic drug induced,occupational/environmental (Farmer lung), granulomatous diseases(sarcoidosis, hypersensitivity pneumonia), collagen vascular disease(scleroderma and others), alveolar proteinosis, langerhans cellgranulonmatosis, lymphangioleiomyomatosis, Hermansky-Pudlak Syndrome,Tuberous sclerosis, neurofibromatosis, metabolic storage disorders,familial interstitial lung disease.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is used to treat post-transplant fibrosisassociated with chronic rejection in a mammal (e.g. Bronchiolitisobliterans for lung transplant).

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is used to treat cutaneous fibrosis in amammal (e.g. cutaneous scleroderma, Dupuytren disease, keloids).

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is used to treat hepatic fibrosis with orwithout cirrhosis in a mammal: toxic/drug induced (hemochromatosis),alcoholic liver disease, viral hepatitis (hepatitis B virus, hepatitis Cvirus, HCV), nonalcoholic liver disease (NASH), metabolic andauto-immune.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to treat renal fibrosis in a mammal:tubulointerstitium fibrosis, glomerular sclerosis.

In any of the aforementioned aspects involving the treatment of LPAdependent diseases or conditions are further embodiments comprisingadministering at least one additional agent in addition to theadministration of Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2). In various embodiments, each agent isadministered in any order, including simultaneously.

In any of the embodiments disclosed herein, the mammal is a human.

In some embodiments, compounds provided herein are administered to ahuman. In some embodiments, compounds provided herein are orallyadministered to a human.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is used for inhibiting the activity of atleast one LPA receptor or for the treatment of a disease or conditionthat would benefit from inhibition of the activity of at least one LPAreceptor. In one aspect, the LPA receptor is LPA₁.

In other embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is used for the formulation of a medicamentfor the inhibition of LPA₁ activity.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is used in the preparation of a medicamentfor use in medicine. In some embodiments, Compound 2, is used in thepreparation of a medicament for use in medicine. In some embodiments,Compound 2, monohydrate is used in the preparation of a medicament foruse in medicine. In some embodiments, Compound 2 (Pattern 1) is used inthe preparation of a medicament for use in medicine.

Also provided is an article of manufacture comprising multiple unitdoses of an oral solid dosage form pharmaceutical composition describedherein in a high-density polyethylene (HDPE) bottle equipped with ahigh-density polyethylene (HDPE) cap.

In some embodiments, high-density polyethylene (HDPE) bottle furthercomprises an aluminum foil induction seal and silica gel desiccant.

In any of the aforementioned embodiments are further embodimentscomprising single administrations of the effective amount of Compound 1,or a pharmaceutically acceptable salt thereof (e.g. Compound 2),including further embodiments in which Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2) is (i) administeredonce-a-day; (ii) is administered twice-a-day; or (iii) is administeredmultiple times over the span of one day.

In any of the aforementioned embodiments are further embodimentscomprising multiple administrations of the effective amount of thecompound, including further embodiments in which (i) the compound isadministered in a single dose; (ii) the time between multipleadministrations is every 6 hours; (iii) the time between multipleadministrations is every 8 hours; (iv) the time between multipleadministrations is every 12 hours.

In some embodiments, the pharmaceutical composition is administereddaily to the mammal.

In some embodiments, the pharmaceutical composition is administered intreatment cycles comprising: (a) a first period during which Compound 2is administered daily to the mammal; and (b) a second period duringwhich the Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2) is administered to the mammal in a reduced amount ascompared to (a) or not administered.

In some embodiments, the methods of treatment or prevention disclosedherein comprise a drug holiday, wherein the administration of Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2) istemporarily suspended or the dose being administered is temporarilyreduced; at the end of the drug holiday dosing is resumed. In someembodiments, the length of the drug holiday varies from 2 days to 1year.

Compound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2) for treating any of the diseases or conditions disclosed herein. Insome embodiments, Compound 1 is crystalline. In some embodiments,Compound 2 is crystalline. In some embodiments, Compound 2 is amorphous.

A pharmaceutical composition comprising Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) for use inany of the uses and methods disclosed herein.

Also described herein are process for the preparation of Compound 1 andpharmaceutically acceptable salts thereof. In one aspect, thepharmaceutically acceptable salt of Compound 1 is the sodium salt(Compound 2).

In one embodiment provided is a process for preparing crystalline1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1) comprising isolating Compound 1 from: ethanol,methanol, 2-methoxyethanol ethanol, 1-propanol, 2-propanol, 1-butanol,butyl acetate, acetone, methylethyl ketone, anisole, toluene,nitromethane, acetonitrile, ethyl acetate, cumene, 1-4-dioxane, ortetrahydrofuran.

In one embodiment provided is a process for preparing crystalline1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt (Compound 2) comprising isolating Compound 2 from:

-   -   (i) methyl ethyl ketone;    -   (ii) methyl ethyl ketone, methyl tert-butyl ether and water;    -   (iii) methyl ethyl ketone, and water;    -   (iv) acetonitrile;    -   (v) 1,4-dioxane and tert-butyl methyl ether;    -   (vi) methyl ethyl ketone and tert-butyl methyl; or    -   (vii) ethanol and heptane.

In one embodiment provided is a process for the preparation of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1) comprising the steps of:

-   -   (1) treatment of a compound of Formula XVIII with        diphenylphosphoryl azide in the presence of        (R)-(+)-1-phenylethanol:

-   -   wherein,    -   R¹ is C₁-C₆alkyl;    -   to provide a compound of Formula X:

-   -   (2) hydrolysis of the ester moiety of the compound of Formula X        to provide Compound 1.

In some embodiments, step (2) comprises treatment of the compound ofFormula X with sodium hydroxide in a suitable solvent followed by a pHadjustment.

In one embodiment provided is a process for the preparation of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1) comprising the steps of:

-   -   (1) reacting a compound of Formula VII:

wherein,

-   -   X is a leaving group;        with a compound of Formula VIII:

-   -   wherein,        -   R¹ is C₁-C₆ alkyl; and B is a boronic acid or boronate            ester,    -   in the presence of a coupling catalyst, a suitable base, and in        a suitable solvent, to provide a compound of Formula X:

-   -   (2) hydrolysis of the ester moiety of the compound of Formula X        to provide Compound 1.

In another embodiment provided is a process for the preparation of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1) comprising the steps of:

-   -   (1) reacting a compound of Formula IX:

-   -   wherein,        -   B is a boronic acid or boronate ester;    -   with a compound of Formula XII:

-   -   wherein,        -   R′ is C₁-C₆ alkyl; and X is a leaving group;    -   in the presence of a coupling catalyst, a suitable base, and in        a suitable solvent, to provide a compound of Formula X:

-   -   (2) hydrolysis of the ester moiety of the compound of Formula X        to provide Compound 1.

In some embodiments, the coupling catalyst is a palladium catalyst. Insome embodiments, the palladium catalyst istetrakis(triphenylphosphine)palladium or(1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II).

In some embodiments, R¹ is —CH₃ or —CH₂CH₃.

In some embodiments, the suitable base is triethylamine,diisopropylethylamine, 1,2,2,6,6-pentamethylpiperidine, tributylamine,sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassiumcarbonate, cesium carbonate, sodium acetate, potassium acetate, sodiumphosphate or potassium phosphate.

In some embodiments, the suitable solvent is tetrahydrofuran, dioxane,water, or combinations thereof.

In some embodiments, X is selected from Cl, Br, I, —OSO₂CF₃,—OSO₂(4-methylphenyl), —OSO₂(phenyl) and —OSO₂CH₃. In some embodiments,X is Br.

In some embodiments, B is

In some embodiments, B is

In some embodiments, B is

In some embodiments, step (1) further comprises isolating the compoundof Formula X prior to step (2).

In some embodiments, step (1) further comprises a purification step forreducing the amount of palladium to less than 20 ppm.

The disclosed processes provide for the synthesis of Compound 1 andpharmaceutically acceptable salts thereof (e.g. Compound 2). Theprocesses disclosed herein are particularly applicable to large scalechemical production of Compound 1 and pharmaceutically acceptable saltsthereof.

In some embodiments, in any of the embodiments disclosed herein(including methods, uses, formulations, combination therapy, etc.),Compound 1, or a pharmaceutically acceptable salt or solvate thereof, isreplaced with: a) Compound 1, or a pharmaceutically acceptable salt orsolvate thereof, of lower chiral purity; b)1-{4′-[3-methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or a pharmaceutically acceptable salt or solvate thereof of anyoptical purity; or c) racemic1-{4′-[3-methyl-4-(1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, or a pharmaceutically acceptable salt or solvate thereof.

In any of the embodiments disclosed herein (including methods, uses,formulations, combination therapy, etc.), amorphous Compound 1 is used.In any of the embodiments disclosed herein (including methods, uses,formulations, combination therapy, etc.), crystalline Compound 1 isused. In any of the embodiments disclosed herein (including methods,uses, formulations, combination therapy, etc.), crystalline Compound 1(Pattern 1) is used. In any of the embodiments disclosed herein(including methods, uses, formulations, combination therapy, etc.),crystalline Compound 1 (Pattern 2) is used. In any of the embodimentsdisclosed herein (including methods, uses, formulations, combinationtherapy, etc.), crystalline Compound 1 (Pattern 3) is used.

In any of the embodiments disclosed herein (including methods, uses,formulations, combination therapy, etc.), amorphous Compound 2 is used.In any of the embodiments disclosed herein (including methods, uses,formulations, combination therapy, etc.), crystalline Compound 2 isused. In any of the embodiments disclosed herein (including methods,uses, formulations, combination therapy, etc.), partially crystallineCompound 2 is used. In any of the embodiments disclosed herein(including methods, uses, formulations, combination therapy, etc.),crystalline Compound 2 (Pattern 1) is used. In any of the embodimentsdisclosed herein (including methods, uses, formulations, combinationtherapy, etc.), crystalline Compound 2 (Pattern 2) is used. In any ofthe embodiments disclosed herein (including methods, uses, formulations,combination therapy, etc.), crystalline Compound 2 (Pattern 3) is used.

In some embodiments, in any of the embodiments disclosed herein(including methods, uses, formulations, combination therapy, etc.),Compound 1, or a pharmaceutically acceptable salt thereof, is replacedwith an active metabolite of Compound 1. In some embodiments, the activemetabolite is in a crystalline form. In some embodiments, the activemetabolite is in an amorphous phase. In some embodiments, in any of theembodiments disclosed herein (including methods, uses, formulations,combination therapy, etc.), Compound 1, or a pharmaceutically acceptablesalt thereof, is replaced with a prodrug of Compound 1, or a deuteratedanalog of Compound 1, or a pharmaceutically acceptable salt thereof.

Other objects, features and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only, since variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the XRPD of Pattern 1 of Crystalline Compound 1.

FIG. 2 illustrates the TGA of Pattern 1 of Crystalline Compound 1.

FIG. 3 illustrates the DSC of Pattern 1 of Crystalline Compound 1.

FIG. 4 illustrates the XRPD of Pattern 1 of Crystalline Compound 2.

FIG. 5 illustrates the TGA of Pattern 1 of Crystalline Compound 2.

FIG. 6 illustrates the DSC of Pattern 1 of Crystalline Compound 2.

FIG. 7 illustrates the IR spectrum of Pattern 1 of Crystalline Compound2.

FIG. 8 illustrates the XRPD of Pattern 2 of Crystalline Compound 2.

FIG. 9 illustrates the XRPD of Pattern 3 of Crystalline Compound 2.

FIG. 10 illustrates the XRPD of amorphous Compound 2.

FIG. 11 illustrates the DSC of amorphous Compound 2.

FIG. 12 illustrates the XRPD of Pattern 2 of Crystalline Compound 1.

FIG. 13 illustrates the XRPD of Pattern 3 of Crystalline Compound 1.

FIG. 14 illustrates the results of Compound 1 on dermal thickness in amouse model of bleomycin-induced scleroderma.

FIG. 15 illustrates the results of Compound 1 on collagen content in amouse model of bleomycin-induced scleroderma.

DETAILED DESCRIPTION OF THE INVENTION

Lysophospholipids (such as lysophosphatidic acid (LPA)) affectfundamental cellular functions that include cellular proliferation,differentiation, survival, migration, adhesion, invasion, andmorphogensis. These functions influence many biological processes thatinclude neurogensis, angiogenesis, wound healing, immunity, andcarcinogenesis.

LPA acts through sets of specific G protein-coupled receptors (GPCRs) inan autocrine and paracrine fashion. LPA binding to its cognate GPCRs(LPA₁, LPA₂, LPA₃, LPA₄, LPA₅, LPA₆) activates intracellular signalingpathways to produce a variety of biological responses.

LPA has a role as a biological effector molecule, and has a diverserange of physiological actions such as, but not limited to, effects onblood pressure, platelet activation, and smooth muscle contraction, anda variety of cellular effects, which include cell growth, cell rounding,neurite retraction, and actin stress fiber formation and cell migration.The effects of LPA are predominantly receptor mediated.

Activation of the LPA receptors with LPA mediates a range of downstreamsignaling cascades. The actual pathway and realized end point aredependent on a range of variables that include receptor usage, celltype, expression level of a receptor or signaling protein, and LPAconcentration. Nearly all mammalian cells, tissues and organs co-expressseveral LPA-receptor subtypes, which indicates that LPA receptors signalin a cooperative manner. LPA₁, LPA₂, and LPA₃ share high amino acidsequence similarity.

LPA regulates many important functions of fibroblasts in wound healing,including proliferation, migration, differentiation and contraction.Fibroblast proliferation is required in wound healing in order to fillan open wound. In contrast, fibrosis is characterized by intenseproliferation and accumulation of myofibroblasts that activelysynthesize ECM and proinflammatory cytokines. LPA can either increase orsuppress the proliferation of cell types important in wound healing.

Tissue injury initiates a complex series of host wound-healingresponses, if successful, these responses restore normal tissuestructure and function. If not, these responses can lead to tissuefibrosis and loss of function.

A number of muscular dystrophics are characterized by a progressiveweakness and wasting of musculature, and by extensive fibrosis. It hasbeen shown that LPA treatment of cultured myoblasts induced significantexpression of connective tissue growth factor (CTGF). CTGF subsequentlyinduces collagen, fibronectin and integrin expression and inducesdedifferentiation of these myoblasts. Treatment of a variety of celltypes with LPA induces reproducible and high level induction of CTGF.CTGF is a profibrotic cytokine, signaling down-stream and in parallelwith TGFβ.

LPA and LPA₁ play key pathogenic roles in pulmonary fibrosis. Fibroblastchemoattractant activity plays an important role in the lungs inpatients with pulmonary fibrosis. Profibrotic effects of LPA₁-receptorstimulation is explained by LPA₁-receptor-mediated vascular leakage andincreased fibroblast recruitment, both profibrotic events. The LPA-LPA₁pathway has a role in mediating fibroblast migration and vascularleakage in IPF. The end result is the aberrant healing process thatcharacterises this fibrotic condition.

The LPA-LPA2 pathway contributes to the activation of the TGF-β pathwayin pulmonary fibrosis. In some embodiments, compounds that inhibit LPA2show efficacy in the treatment of lung fibrosis. In some embodiments,compounds that inhibit both LPA1 and LPA2 show improved efficacy in thetreatment of lung fibrosis compared to compounds which inhibit only LPA1or LPA2.

LPA and LPA₁ are involved in the etiology of kidney fibrosis. In miceinvalidated for the LPA₁ receptor (LPA₁ (−/−), the development of renalfibrosis was significantly attenuated. Unilateral ureteral obstruction(UUO; animal model of renal fibrosis) mice treated with the LPA receptorantagonist Ki16425 closely resembled the LPA₁ (−/−) mice.

LPA is implicated in liver disease and fibrosis. Plasma LPA levels andserum autotoxin are elevated in hepatitis patients and animal models ofliver injury in correlation with increased fibrosis. LPA also regulatesliver cell function. LPA₁ and LPA₂ receptors are expressed by mousehepatic stellate cells and LPA stimulates migration of hepaticmyofibroblasts.

LPA is in involved in wound healing in the eye. LPA₁ and LPA₃ receptorsare detectable in the normal rabbit corneal epithelial cells,keratocytes and endothelial cells and LPA₁ and LPA₃ expression areincreased in corneal epithelial cells following injury.

LPA is present in the aqueous humor and the lacrimal gland fluid of therabbit eye and these levels are increased in a rabbit corneal injurymodel.

LPA induces actin stress fiber formation in rabbit corneal endothelialand epithelial cells and promotes contraction corneal fibroblasts. LPAalso stimulates proliferation of human retinal pigmented epithelialcells.

LPA is implicated in myocardial infarction and cardiac fibrosis. SerumLPA levels are increased in patients following mycocardial infarction(MI) and LPA stimulates proliferation and collagen production (fibrosis)by rat cardiac fibroblasts. Both LPA1 and LPA3 receptors are highlyexpressed in human heart tissue.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to treat or prevent fibrosis in a mammal. Inone aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to treat or prevent fibrosis of an organ ortissue in a mammal.

The terms “fibrosis” or “fibrosing disorder,” as used herein, refers toconditions that are associated with the abnormal accumulation of cellsand/or fibronectin and/or collagen and/or increased fibroblastrecruitment and include but are not limited to fibrosis of individualorgans or tissues such as the heart, kidney, liver, joints, lung,pleural tissue, peritoneal tissue, skin, cornea, retina, musculoskeletaland digestive tract.

Exemplary diseases, disorders, or conditions that involve fibrosisinclude, but are not limited to: lung diseases associated with fibrosis,e.g., idiopathic pulmonary fibrosis, pulmonary fibrosis secondary tosystemic inflammatory disease such as rheumatoid arthritis, scleroderma,lupus, cryptogenic fibrosing alveolitis, radiation induced fibrosis,chronic obstructive pulmonary disease (COPD), chronic asthma, silicosis,asbestos induced pulmonary or pleural fibrosis, acute lung injury andacute respiratory distress (including bacterial pneumonia induced,trauma induced, viral pneumonia induced, ventilator induced,non-pulmonary sepsis induced, and aspiration induced); Chronicnephropathies associated with injury/fibrosis (kidney fibrosis), e.g.,glomerulonephritis secondary to systemic inflammatory diseases such aslupus and scleroderma, diabetes, glomerular nephritis, focal segmentalglomerular sclerosis, IgA nephropathy, hypertension, allograft andAlport; Gut fibrosis, e.g., scleroderma, and radiation induced gutfibrosis; Liver fibrosis, e.g., cirrhosis, alcohol induced liverfibrosis, nonalcoholic steatohepatitis (NASH), biliary duct injury,primary biliary cirrhosis, infection or viral induced liver fibrosis(e.g., chronic HCV infection), and autoimmune hepatitis; Head and neckfibrosis, e.g., radiation induced; Corneal scarring, e.g., LASIK(laser-assisted in situ keratomileusis), corneal transplant, andtrabeculectomy; Hypertrophic scarring and keloids, e.g., burn induced orsurgical; and other fibrotic diseases, e.g., sarcoidosis, scleroderma,spinal cord injury/fibrosis, myelofibrosis, vascular restenosis,atherosclerosis, arteriosclerosis, Wegener's granulomatosis, mixedconnective tissue disease, and Peyronie's disease.

In one aspect, a mammal suffering from one of the following non-limitingexemplary diseases, disorders, or conditions will benefit from therapywith Compound 1, or a pharmaceutically acceptable salt thereof (e.g.Compound 2): atherosclerosis, thrombosis, heart disease, vasculitis,formation of scar tissue, restenosis, phlobitis, COPD (chronicobstructive pulmonary disease), pulmonary hypertension, pulmonaryfibrosis, pulmonary inflammation, bowel adhesions, bladder fibrosis andcystitis, fibrosis of the nasal passages, sinusitis, inflammationmediated by neutrophils, and fibrosis mediated by fibroblasts.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to treat a dermatological disorders in amammal. Dermatological disorders include, but are not limited to,proliferative or inflammatory disorders of the skin such as, atopicdermatitis, bullous disorders, collagenoses, psoriasis, psoriaticlesions, scleroderma, dermatitis, contact dermatitis, eczema, urticaria,rosacca, scleroderma, wound healing, scarring, hypertrophic scarring,keloids, Kawasaki Disease, rosacea. Sjogren-Larsso Syndrome, urticaria.

LPA is released following tissue injury. LPA₁ plays a role in theinitiation of neuropathic pain. In one aspect, Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), is used inthe treatment of pain in a mammal. In one aspect, the pain is acute painor chronic pain. In another aspect, the pain is neuropathic pain. Inanother aspect, the pain is cancer pain. In one aspect, Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), is used inthe treatment of fibromylagia.

Lysophospholipid receptor signaling plays a role in the etiology ofcancer. Lysophosphatidic acid (LPA) and its G protein-coupled receptors(GPCRs) LPA₁, LPA₂, and/or LPA₃ play a role in the development ofseveral types of cancers.

LPA contributes to tumorigenesis by increasing motility and invasivenessof cells. LPA has been implicated in the initiation or progression ofovarian cancer. LPA is present at significant concentrations (2-80 μM)in the ascitic fluid of ovarian cancer patients. LPA receptors (LPA2 andLPA3) are also overexpressed in ovarian cancer cells as compared tonormal ovarian surface epithelial cells. LPA has also been implicated inthe initiation or progression of prostate cancer, breast cancer,melanoma, head and neck cancer, bowel cancer (colorectal cancer),thyroid cancer, glioblastoma, and other cancers.

LPA receptors mediate both migration of and invasion by pancreaticcancer cell lines: Ki16425 and LPA1-specific siRNA effectively blockedin vitro migration in response to LPA and peritoneal fluid (ascites)from pancreatic cancer patients; in addition. Ki16425 blocked theLPA-induced and ascites-induced invasion activity of a highly peritonealmetastatic pancreatic cancer cell line (Yamada et al, J. Biol. Chem.,279, 6595-6605, 2004).

Colorectal carcinoma cell lines show significant expression of LPA₁ mRNAand respond to LPA by cell migration and production of angiogenicfactors. Overexpression of LPA receptors has a role in the pathogenesisof thyroid cancer. LPA₃ was originally cloned from prostate cancercells, concordant with the ability of LPA to induce autocrineproliferation of prostate cancer cells.

LPA has stimulatory roles in cancer progression in many types of cancer.LPA is produced from and induces proliferation of prostate cancer celllines. LPA induces human colon carcinoma DLD1 cell proliferation,migration, adhesion, and secretion of angiogenic factors through LPA₁signalling. In other human colon carcinoma cells lines (HT29 and WiDR),LPA enhances cell proliferation and secretion of angiogenic factors. Inother colon cancer cell lines, LPA₂ and LPA₃ receptor activation resultsin proliferation of the cells. LPA₁ is implicated in bone metastasis(Boucharaba et al., Proc. Natl. Acad. Sci USA, 103, 9643-9648, 2006).

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used in the treatment of cancer. In one aspect,Compound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2), is used in the treatment of malignant and benign proliferativedisease. In one aspect, Compound 1, or a pharmaceutically acceptablesalt thereof (e.g. Compound 2), is used to prevent or reduceproliferation of tumor cells, invasion and metastasis of carcinomas,pleural mesothelioma or peritoneal mesothelioma, cancer pain, bonemetastases. In one aspect is a method of treating cancer in a mammal,the method comprising administering to the mammal Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), and a secondtherapeutic agent, wherein the second therapeutic agent is ananti-cancer agent. In some embodiments, radiation therapy is also used.

The types of cancer include, but is not limited to, solid tumors (suchas those of the bladder, bowel, brain, breast, endometrium, heart,kidney, lung, lymphatic tissue (lymphoma), ovary, pancreas or otherendocrine organ (thyroid), prostate, skin (melanoma or basal cellcancer) or hematological tumors (such as the leukemias) at any stage ofthe disease with or without metastases.

Additional non-limiting examples of cancers include, acute lymphoblasticleukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer,appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer,bladder cancer, bone cancer (osteosarcoma and malignant fibroushistiocytoma), brain stem glioma, brain tumors, brain and spinal cordtumors, breast cancer, bronchial tumors, Burkitt lymphoma, cervicalcancer, chronic lymphocytic leukemia, chronic myelogenous leukemia,colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-Celllymphoma, embryonal tumors, endometrial cancer, ependymoblastoma,ependymoma, esophageal cancer, ewing sarcoma family of tumors, eyecancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),gastrointestinal stromal cell tumor, germ cell tumor, glioma, hairy cellleukemia, head and neck cancer, hepatocellular (liver) cancer, hodgkinlymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors(endocrine pancreas), Kaposi sarcoma, kidney cancer, Langerhans cellhistiocytosis, laryngeal cancer, leukemia, Acute lymphoblastic leukemia,acute myeloid leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, hairy cell leukemia, liver cancer, non-small celllung cancer, small cell lung cancer, Burkitt lymphoma, cutaneous T-celllymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, lymphoma, Waldenströmmacroglobulinemia, medulloblastoma, medulloepithelioma, melanoma,mesothelioma, mouth cancer, chronic myelogenous leukemia, myeloidleukemia, multiple myeloma, nasopharyngeal cancer, neuroblastoma,non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer,oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytoma ofbone, ovarian cancer, ovarian epithelial cancer, ovarian germ celltumor, ovarian low malignant potential tumor, pancreatic cancer,papillomatosis, parathyroid cancer, penile cancer, pharyngeal cancer,pineal parenchymal tumors of intermediate differentiation, pineoblastomaand supratentorial primitive neuroectodermal tumors, pituitary tumor,plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primarycentral nervous system lymphoma, prostate cancer, rectal cancer, renalcell (kidney) cancer, retinoblastoma, rhabdomyosarcoma, salivary glandcancer, sarcoma, Ewing sarcoma family of tumors, sarcoma, kaposi, Sézarysyndrome, skin cancer, small cell Lung cancer, small intestine cancer,soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer,supratentorial primitive neuroectodermal tumors, T-cell lymphoma,testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroidcancer, urethral cancer, uterine cancer, uterine sarcoma, vaginalcancer, vulvar cancer, Waldenström macroglobulinemia, Wilms tumor.

In one aspect, LPA is a contributor to the pathogenesis of respiratorydiseases. Proinflammatory effects of LPA include degranulation of mastcells, contraction of smooth-muscle cells and release of cytokines fromdendritic cells. LPA induces the secretion of IL-8 from human bronchialepithelial cells. IL-8 is found in increased concentrations in BALfluids from patients with asthma, chronic obstructive lung disease,pulmonary sarcoidosis and acute respiratory distress syndrome and Il-8has been shown to exacerbate airway inflammation and airway remodelingof asthmatics. LPA1, LPA2 and LPA3 receptors have all been shown tocontribute to the LPA-induced IL-8 production.

Administration of LPA in vivo induces airway hyper-responsiveness,itch-scratch responses, infiltration and activation of eosinophils andneutrophils, vascular remodeling, and nociceptive flexor responses. LPAalso induces histamine release from mouse and rat mast cells. In oneaspect, the effects of LPA are mediated through LPA₁ and/or LPA₃. In oneaspect, Compound 1, or a pharmaceutically acceptable salt thereof (e.g.Compound 2), is used in the treatment of various allergic disorders in amammal. In one aspect, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is used in the treatment of respiratorydiseases, disorders or conditions in a mammal. In one aspect, Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2), isused in the treatment of asthma in a mammal. In one aspect, Compound 1,or a pharmaceutically acceptable salt thereof (e.g. Compound 2), is usedin the treatment of chronic asthma in a mammal.

The term “respiratory disease,” as used herein, refers to diseasesaffecting the organs that are involved in breathing, such as the nose,throat, larynx, eustachian tubes, trachea, bronchi, lungs, relatedmuscles (e.g., diaphram and intcrcostals), and nerves. Respiratorydiseases include, but are not limited to, asthma, adult respiratorydistress syndrome and allergic (extrinsic) asthma, non-allergic(intrinsic) asthma, acute severe asthma, chronic asthma, clinicalasthma, nocturnal asthma, allergen-induced asthma, aspirin-sensitiveasthma, exercise-induced asthma, isocapnic hyperventilation, child-onsetasthma, adult-onset asthma, cough-variant asthma, occupational asthma,steroid-resistant asthma, seasonal asthma, seasonal allergic rhinitis,perennial allergic rhinitis, chronic obstructive pulmonary disease,including chronic bronchitis or emphysema, pulmonary hypertension,interstitial lung fibrosis and/or airway inflammation and cysticfibrosis, and hypoxia.

In one aspect, presented herein is the use of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), in thetreatment or prevention of chronic obstructive pulmonary disease in amammal comprising administering to the mammal at least once an effectiveamount of Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2). In addition, chronic obstructive pulmonary diseaseincludes, but is not limited to, chronic bronchitis or emphysema,pulmonary hypertension, interstitial lung fibrosis and/or airwayinflammation, and cystic fibrosis.

The nervous system is a major locus for LPA₁ expression. In one aspect,provided is Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), for use in the treatment or prevention of a nervoussystem disorder in a mammal. The term “nervous system disorder,” as usedherein includes, but is not limited to, Alzheimer's Disease, cerebraledema, cerebral ischemia, stroke, multiple sclerosis, neuropathies,Parkinson's Disease, multiple sclerosis, retinal ischemia, post-surgicalcognitive dysfunction, migraine, peripheral neuropathy/neuropathic pain,spinal cord injury, cerebral edema and head injury.

Angiogenesis, the formation of new capillary networks from pre-existingvasculature, is normally invoked in wound healing, tissue growth andmyocardial angiogenesis after ischemic injury. Peptide growth factorsand lysophospholipids control coordinated proliferation, migration,adhesion, differentiation and assembly of vascular endothelial cells(VECs) and surrounding vascular smooth-muscle cells (VSMCs). In oneaspect, dysregulation of the processes mediating angiogenesis leads toatherosclerosis, hypertension, tumor growth, rheumatoid arthritis anddiabetic retinopathy.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to treat or prevent cardiovascular disease inmammal, including but not limited to: arrhythmia (atrial or ventricularor both); atherosclerosis and its sequalae; angina; cardiac rhythmdisturbances; myocardial ischemia; myocardial infarction; cardiac orvascular aneurysm; vasculitis, stroke; peripheral obstructivearteriopathy of a limb, an organ, or a tissue; reperfusion injuryfollowing ischemia of the brain, heart, kidney or other organ or tissue;endotoxic, surgical, or traumatic shock; hypertension, valvular heartdisease, heart failure, abnormal blood pressure; shock; vasoconstriction(including that associated with migraines); vascular abnormality,inflammation, insufficiency limited to a single organ or tissue.

In one aspect, provided herein are methods for preventing or treatingvasoconstriction, atherosclerosis and its sequelae myocardial ischemia,myocardial infarction, aortic aneurysm, vasculitis and stroke comprisingadministering at least once to the mammal an effective amount ofCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2). In some embodiments, the LPA antagonist disclosed herein is used totreat Raynaud's phenomenon. Raynaud's phenomenon comprises bothRaynaud's disease (where the phenomenon is idiopathic) and Raynaud'ssyndrome, where it is caused by some other instigating factor.

In one aspect, provided herein are methods for reducing cardiacreperfusion injury following myocardial ischemia and/or endotoxic shockcomprising administering at least once to the mammal an effective amountof Compound 1, or a pharmaceutically acceptable salt thereof (e.g.Compound 2).

In one aspect, provided herein are methods for reducing the constrictionof blood vessels in a mammal comprising administering at least once tothe mammal an effective amount of Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2).

In one aspect, provided herein are methods for lowering or preventing anincrease in blood pressure of a mammal comprising administering at leastonce to the mammal an effective amount of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2).

LPA is associated with various inflammatory/immune diseases. In oneaspect, Compound 1, or a pharmaceutically acceptable salt thereof (e.g.Compound 2), is used to treat or prevent inflammation in a mammal. Inone aspect, antagonists of LPA₁ and/or LPA₃ find use in the treatment orprevention of inflammatory/immune disorders in a mammal.

Examples of inflammatory/immune disorders include psoriasis, rheumatoidarthritis, vasculitis, inflammatory bowel disease, dermatitis,osteoarthritis, asthma, inflammatory muscle disease, allergic rhinitis,vaginitis, interstitial cystitis, scleroderma, eczema, allogeneic orxenogeneic transplantation (organ, bone marrow, stem cells and othercells and tissues) graft rejection, graft-versus-host disease, lupuserythematosus, inflammatory disease, type I diabetes, pulmonaryfibrosis, dermatomyositis, Sjogren's syndrome, thyroiditis (e.g.,Hashimoto's and autoimmune thyroiditis), myasthenia gravis, autoimmunehemolytic anemia, multiple sclerosis, cystic fibrosis, chronic relapsinghepatitis, primary biliary cirrhosis, allergic conjunctivitis and atopicdermatitis.

In accordance with one aspect, are methods for treating, preventing,reversing, halting or slowing the progression of LPA-dependent orLPA-mediated diseases or conditions once it becomes clinically evident,or treating the symptoms associated with or related to LPA-dependent orLPA-mediated diseases or conditions, by administering to the mammalCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2). In certain embodiments, the subject already has a LPA-dependent orLPA-mediated disease or condition at the time of administration, or isat risk of developing a LPA-dependent or LPA-mediated disease orcondition.

In certain aspects, are methods for preventing or treating eosinophiland/or basophil and/or dendritic cell and/or neutrophil and/or monocyteand/or T-cell recruitment comprising administering at least once to themammal an effective amount of Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2).

In certain aspects, are methods for the treatment of cystitis,including, e.g., interstitial cystitis, comprising administering atleast once to the mammal a therapeutically effective amount of Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2).

In accordance with one aspect, methods described herein include thediagnosis or determination of whether or not a patient is suffering froma LPA-dependent or LPA-mediated disease or condition by administering tothe subject a therapeutically effective amount of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), anddetermining whether or not the patient responds to the treatment.

In one aspect provided herein is Compound 1, pharmaceutically acceptablesalts, pharmaceutically acceptable prodrugs, and pharmaceuticallyacceptable solvates thereof, which is an antagonist of at least one LPAreceptor (e.g. LPA₁, LPA₂, LPA₃) and is used to treat patients sufferingfrom one or more LPA-dependent or LPA-mediated conditions or diseases,including, but not limited to, lung fibrosis, kindney fibrosis, liverfibrosis, scarring, scleroderma, asthma, rhinitis, chronic obstructivepulmonary disease, pulmonary hypertension, interstitial lung fibrosis,arthritis, allergy, psoriasis, inflammatory bowel disease, adultrespiratory distress syndrome, myocardial infarction, aneurysm, stroke,cancer, pain, proliferative disorders and inflammatory conditions. Insome embodiments, LPA-dependent conditions or diseases include thosewherein an absolute or relative excess of LPA is present and/orobserved.

In any of the aforementioned aspects the LPA-dependent or LPA-mediateddiseases or conditions include, but are not limited to, organ fibrosis,tissue fibrosis, asthma, allergic disorders, chronic obstructivepulmonary disease, pulmonary hypertension, lung or pleural fibrosis,peritoneal fibrosis, arthritis, allergy, cancer, cardiovascular disease,adult respiratory distress syndrome, myocardial infarction, aneurysm,stroke, and cancer.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to improve the corneal sensitivity decreasecaused by corneal operations such as laser-assisted in situkeratomileusis (LASIK) or cataract operation, corneal sensitivitydecrease caused by corneal degeneration, and dry eye symptom causedthereby.

In one aspect, presented herein is the use of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), in thetreatment or prevention of ocular inflammation and allergicconjunctivitis, vernal keratoconjunctivitis, and papillaryconjunctivitis in a mammal.

In one aspect, presented herein is the use of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), in thetreatment or prevention of Sjogren disease or inflammatory disease withdry eyes in a mammal.

In one aspect, LPA and LPA receptors (e.g. LPA₁) are involved in thepathogenesis of osteoarthritis. In one aspect, presented herein is theuse of Compound 1, or a pharmaceutically acceptable salt thereof (e.g.Compound 2), in the treatment or prevention of osteoarthritis in amammal.

In one aspect, LPA receptors (e.g. LPA₁, LPA₃) contribute to thepathogenesis of rheumatoid arthritis. In one aspect, presented herein isthe use of Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), in the treatment or prevention of rheumatoidarthritis in a mammal.

In one aspect, LPA receptors (e.g. LPA₁) contribute to adipogenesis. Inone aspect, presented herein is the use of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), in thepromotion of adipose tissue formation in a mammal.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is used to treat Raynaud's phenomenon in a mammal.Raynaud's phenomenon comprises both Raynaud's disease (where thephenomenon is idiopathic) and Raynaud's syndrome, where it is caused bysome instigating factor.

Described herein are compositions, pharmaceutical compositions, methodsfor treating, methods for formulating, methods for producing, methodsfor manufacturing, treatment strategies, pharmacokinetic strategiesusing Compound 1, or pharmaceutically acceptable salts thereof.

Compound 1, and Pharmaceutically Acceptable Salts Thereof

“Compound 1” or“1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid,” “(R)-1-phenylethyl-5-(4-biphenyl-4-cyclopropanecarboxylicacid)-3-methylisoxazole-4-yl carbamate” or any other similar name refersto the compound with the following structure:

In some embodiments, Compound 1 is substantially free of the S-isomer.

“Substantially free” with respect to an enantiomer, means that thereferenced enantiomer is not present or there is less than 5%, less than4%, less than 3%, less than 2% or less than 1% of the referencedenantiomer.

“Compound 2” or“1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid, sodium salt” or “sodium1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylate”or “(R)-1-phenylethyl-5-(4-biphenyl-4-cyclopropanecarboxylicacid)-3-methylisoxazole-4-yl carbamate sodium salt” or any other similarname refers to the compound with the following structure:

In some embodiments, Compound 2 is substantially free of the S-isomer.

A wide variety of pharmaceutically acceptable salts are formed fromCompound 1 and include:

-   -   salts formed when the acidic proton of the carboxylic acid of        Compound 1 is replaced by a metal ion, such as for example, an        alkali metal ion (e.g. lithium, sodium, potassium), an alkaline        earth ion (e.g. magnesium, or calcium), or an aluminum ion, or        is replaced by an ammonium cation (NH₄ ⁺);    -   salts formed by reacting Compound 1 with a pharmaceutically        acceptable organic base, which includes alkylamines, such as        choline, ethanolamine, diethanolamine, triethanolamine,        tromethamine, N-methylglucamine, dicyclohexylamine,        tris(hydroxymethyl)methylamine, and salts with amino acids, such        as arginine, lysine, and the like.

In some embodiments, Compound 1 is treated with an amino acid to form asalt.

In other embodiments, Compound 1 is treated with choline, ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine,arginine, lysine, ammonium hydroide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like to form asalt.

The term “pharmaceutically acceptable salt” in reference to Compound 1refers to a salt of Compound 1, which does not cause significantirritation to a mammal to which it is administered and does notsubstantially abrogate the biological activity and properties of thecompound. In some embodiments, the pharmaceutically acceptable salt ofCompound 1 is a lithium salt, sodium salt, potassium salt, magnesiumsalt, calcium salt, ammonium salt, choline salt, ethanolamine salt,diethanolamine salt, triethanolamine salt, tromethamine salt,N-methylglucamine salt, dicyclohexylamine salt,tris(hydroxymethyl)methylamine salt, arginine salt, or lysine salt. Insome embodiments, the pharmaceutically acceptable salt of Compound 1 isa sodium salt.

It should be understood that a reference to a pharmaceuticallyacceptable salt includes the solvent addition forms (solvates). Solvatescontain either stoichiometric or non-stoichiometric amounts of asolvent, and are formed during the process of product formation orisolation with pharmaceutically acceptable solvents such as water,ethanol, methyl tert-butyl ether, isopropanol, acetonitrile, heptane,and the like. In one aspect, solvates are formed using, but not limitedto, Class 3 solvent(s). Categories of solvents are defined in, forexample, the International Conference on Harmonization of TechnicalRequirements for Registration of Pharmaceuticals for Human Use (ICH),“Impurities: Guidelines for Residual Solvents, Q3C(R3), (November 2005).Hydrates are formed when the solvent is water, or alcoholates are formedwhen the solvent is alcohol. In one embodiment, solvates of Compound 1,or pharmaceutically acceptable salts thereof, are conveniently preparedor formed during the processes described herein. In addition, Compound1, or pharmaceutically acceptable salts thereof, exist in unsolvatedform. In some embodiments, Compound 1, or a pharmaceutically acceptablesalt thereof, is hydrated. In some embodiments, Compound 2 is hydrated.In some embodiments, Compound 2 is a monohydrate.

In yet other embodiments. Compound 1, or a pharmaceutically acceptablesalt thereof (e.g. Compound 2) is prepared in various forms, includingbut not limited to, amorphous phase, milled forms and nano-particulateforms.

Amorphous Compound 1

In some embodiments, Compound 1 is amorphous. In some embodiments,Amorphous Phase of Compound 1 has an XRPD pattern showing a lack ofcrystallinity.

Compound 1—Pattern 1

In some embodiments, Compound 1 is crystalline. In some embodiments,Compound 1 is crystalline Pattern 1. Crystalline Pattern 1 of Compound 1is characterized as having:

-   -   (a) an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 4.7° 2-Theta, 9.4° 2-Theta, 14.50°        2-Theta, and 21.0° 2-Theta;    -   (b) an X-ray powder diffraction (XRPD) pattern substantially the        same as the XRPD shown in FIG. 1;    -   (c) a DSC thermogram with an endotherm at about 172-176° C.;    -   (d) a DSC or a thermo-gravimetric analysis (TGA) substantially        similar to the ones set forth in FIG. 2 and FIG. 3;    -   (e) substantially the same X-ray powder diffraction (XRPD)        pattern post storage at 40 C/75% relative humidity for one week;    -   (f) unit cell parameters substantially equal to the following at        25° C.:

a (Å) 26.2070(8) b (Å) 37.700(1) c (Å) 5.0051(2) α° 90 β° 90 γ° 90 V(Å3) 4945.1(3) Z 8 Calculated Density 1.296 Crystal System OrthorhombicSG P2₁2₁2 R1 0.0418 Sol. Sites —

-   -   (g) combinations thereof.

In some embodiments, the crystalline form of Compound 1 is substantiallyfree of the S-isomer. In some embodiments, the crystalline form ofCompound 1 is substantially free of amorphous Compound 1.

In some embodiments, the crystalline form of Compound 1 is crystallizedfrom ethanol, methanol, 2-methoxyethanol, ethanol, 1-propanol,2-propanol, 1-butanol, butyl acetate, acetone, methylethyl ketone,anisole, toluene, nitromethane, acetonitrile, ethyl acetate, cumene,1-4-dioxane, tetrahydrofuran, dichloromethane, heptane, or combinationsthereof.

In some embodiments, Crystalline Pattern 1 of Compound 1 ischaracterized as having at least one of the properties selected from (a)to (f). In some embodiments, Crystalline Pattern 1 of Compound 1 ischaracterized as having at least two of the properties selected from (a)to (f). In some embodiments, Crystalline Pattern 1 of Compound 1 ischaracterized as having at least three of the properties selected from(a) to (f). In some embodiments, Crystalline Pattern 1 of Compound 1 ischaracterized as having at least four of the properties selected from(a) to (f). In some embodiments, Crystalline Pattern 1 of Compound 1 ischaracterized as having at least five of the properties selected from(a) to (f). In some embodiments, Crystalline Pattern 1 of Compound 1 ischaracterized as having the properties (a), (b), (c), (d), (e), and (f).

In some embodiments, crystalline Compound 1 has an X-ray powderdiffraction (XRPD) pattern with characteristic peaks at 4.7° 2-Theta,9.4° 2-Theta, 14.5° 2-Theta, and 21.0° 2-Theta.

In some embodiments, crystalline Compound 1 has an X-ray powderdiffraction (XRPD) pattern substantially the same as the XRPD shown inFIG. 1.

In some embodiments, crystalline Compound 1 has a DSC thermogram with anendotherm at about 172°-176° C.

In some embodiments, crystalline Compound 1 has a DSC or athermo-gravimetric analysis (TGA) substantially similar to the ones setforth in FIG. 2 or FIG. 3.

In some embodiments, crystalline Compound 1 has substantially the sameX-ray powder diffraction (XRPD) pattern post storage at 40 C/75%relative humidity for one week.

In one embodiment, Crystalline Pattern 1 of Compound 1 is characterizedby unit cell parameters approximately equal to the following at atemperature of 25° C.:

a (Å) 26.2070(8) b (Å) 37.700(1) c (Å) 5.0051(2) α° 90 β° 90 γ° 90 V(Å3) 4945.1(3) Z 8 Calculated Density 1.296 Crystal System OrthorhombicSG P2₁2₁2 R1 0.0418 Sol. Sites —

In a further embodiment, Crystalline Pattern 1 of Compound 1 ischaracterized by fractional atomic coordinates substantially the same aslisted in Table 2.

Compound 1—Pattern 2

In some embodiments, Compound 1 is crystalline. In some embodiments,Compound 1 is crystalline Pattern 2. Crystalline Pattern 2 of Compound 1is characterized as having:

-   -   (a) an X-ray powder diffraction (XRPD) pattern substantially the        same as the XRPD shown in FIG. 12;    -   (b) an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 6.3° 2-Theta, 12.8° 2-Theta, 16.4°        2-Theta, 17.0° 2-Theta, and 19.7° 2-Theta;    -   (c) unit cell parameters approximately equal to the following at        a temperature of 25° C.:

a (Å) 30.3522(9) b (Å) 7.8514(3) c (Å) 22.4570(7) α° 90 β° 111.665(2) γ°90 V (Å³) 4973.6(3) Z 8 Calculated Density 1.289 Crystal SystemMonoclinic SG C2 R1 0.0298 Sol. Sites —

-   -   or    -   (d) combinations thereof.

In some embodiments, Crystalline Pattern 2 of Compound 1 ischaracterized as having at least one of the properties selected from (a)to (c). In some embodiments, Crystalline Pattern 2 of Compound 1 ischaracterized as having at least two of the properties selected from (a)to (c). In some embodiments, Crystalline Pattern 2 of Compound 1 ischaracterized as having properties (a), (b), and (c).

In some embodiments, the crystalline form of Compound 1 is substantiallyfree of the S-isomer. In some embodiments, the crystalline form ofCompound 1 is substantially free of amorphous Compound 1. In someembodiments, the crystalline form of Compound 1 has an X-ray powderdiffraction (XRPD) pattern substantially the same as the XRPD shown inFIG. 12. In some embodiments, the crystalline form of Compound 1 has anX-ray powder diffraction (XRPD) pattern with characteristic peaks at6.3° 2-Theta, 12.8° 2-Theta, 16.4° 2-Theta, 17.0° 2-Theta, and 19.7°2-Theta.

In one embodiment, Crystalline Pattern 2 of Compound 1 is characterizedby unit cell parameters approximately equal to the following at atemperature of 25° C.:

a (Å) 30.3522(9) b (Å) 7.8514(3) c (Å) 22.4570(7) α° 90 β° 111.665(2) γ°90 V (Å³) 4973.6(3) Z 8 Calculated Density 1.289 Crystal SystemMonoclinic SG C2 R1 0.0298 Sol. Sites —

In a further embodiment, Crystalline Pattern 2 of Compound 1 ischaracterized by fractional atomic coordinates substantially the same aslisted in Table 4.

Compound 1—Pattern 3

In some embodiments, Compound 1 is crystalline. In some embodiments,Compound 1 is crystalline Pattern 3. Crystalline Pattern 3 of Compound 1is characterized as having:

-   -   (a) an X-ray powder diffraction (XRPD) pattern substantially the        same as the XRPD shown in FIG. 13;    -   (b) an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 5.5° 2-Theta, 5.9° 2-Theta, 12.6°        2-Theta, and 16.7° 2-Theta;    -   (c) unit cell parameters approximately equal to the following at        a temperature of 25° C.:

a (Å) 32.3574(9) b (Å) 5.1057(2) c (Å) 33.148(1) α° 90 β° 114.846(2) γ°90 V (Å³) 4969.4(3) Z 8 Calculated Density 1.290 Crystal SystemMonoclinic SG C2 R1 0.0553 Sol. Sites —

-   -   or    -   (d) combinations thereof.

In some embodiments, Crystalline Pattern 3 of Compound 1 ischaracterized as having at least one of the properties selected from (a)to (c). In some embodiments, Crystalline Pattern 3 of Compound 1 ischaracterized as having at least two of the properties selected from (a)to (c). In some embodiments, Crystalline Pattern 3 of Compound 1 ischaracterized as having properties (a), (b), and (c).

In some embodiments, the crystalline form of Compound 1 is substantiallyfree of the S-isomer. In some embodiments, the crystalline form ofCompound 1 is substantially free of amorphous Compound 1. In someembodiments, the crystalline form of Compound 1 has an X-ray powderdiffraction (XRPD) pattern substantially the same as the XRPD shown inFIG. 13. In some embodiments, the crystalline form of Compound 1 has anX-ray powder diffraction (XRPD) pattern with characteristic peaks at5.5° 2-Theta, 5.9° 2-Theta, 12.6° 2-Theta, 16.7° 2-Theta.

In one embodiment, Crystalline Pattern 3 of Compound 1 is characterizedby unit cell parameters approximately equal to the following at atemperature of 25° C.:

a (Å) 32.3574(9) b (Å) 5.1057(2) c (Å) 33.148(1) α° 90 β° 114.846(2) γ°90 V (Å³) 4969.4(3) Z 8 Calculated Density 1.290 Crystal SystemMonoclinic SG C2 R1 0.0553 Sol. Sites —

In a further embodiment, Crystalline Pattern 3 of Compound 1 ischaracterized by fractional atomic coordinates substantially the same aslisted in Table 6.

Amorphous Compound 2

In some embodiments, Compound 2 is amorphous. In some embodiments,Amorphous Phase of Compound 2 has an XRPD pattern showing a lack ofcrystallinity. In some embodiments, Compound 2 is amorphous and has anX-ray powder diffraction (XRPD) pattern substantially the same as shownin FIG. 10. In some embodiments, Compound 2 is amorphous and has a DSCsubstantially similar to the one set forth in FIG. 11.

Compound 2—Pattern 1

In some embodiments, Compound 2 is crystalline. In some embodiments,Compound 2 is crystalline and hydrated. In some embodiments, Compound 2is crystalline Pattern 1.

In some embodiments, described is a hydrated crystalline form ofCompound 2 (Pattern 1), wherein the hydrated crystalline form ofCompound 2:

-   -   (a) has an X-ray powder diffraction (XRPD) pattern with        characteristic peaks at 13.2° 2-Theta, 17.2° 2-Theta, 19.3°        2-Theta, 22.4° 2-Theta, and 25.6° 2-Theta;    -   (b) has an X-ray powder diffraction (XRPD) pattern substantially        the same as shown in FIG. 4;    -   (c) has a thermo-gravimetric analysis (TGA) or a DSC        substantially similar to the ones set forth in FIG. 5 and FIG.        6;    -   (d) has an infrared spectrum substantially similar to the one        set forth in FIG. 7;    -   (e) was obtained from methyl ethyl ketone, acetonitrile,        1,4-dioxane/tert-butyl methyl ether, methyl ethyl ketone        (MEK)/tert-butyl methyl, or ethanol/heptane;    -   (f) has unit cell parameters substantially equal to the        following at 25° C.:

a(Å) 13.8714(2) b(Å) 7.7379(2) c(Å) 25.5253(5) α ° 90 β° 103.863(1) γ °90 V(Å3) 2659.96(9) Z 4 Calculated Density 1.305 Crystal SystemMonoclinic SG P2₁ R1 0.0301 Sol. Sites 1H₂O

-   -   or    -   (g) combinations thereof.

In some embodiments, the hydrated crystalline form of Compound 2(Pattern 1) is characterized as having at least one property selectedfrom (a) to (f). In some embodiments, the hydrated crystalline form ofCompound 2 (Pattern 1) is characterized as having at least two of theproperties selected from (a) to (f). In some embodiments, the hydratedcrystalline form of Compound 2 (Pattern 1) is characterized as having atleast three of the properties selected from (a) to (f). In someembodiments, the hydrated crystalline form of Compound 2 (Pattern 1) ischaracterized as having at least three of the properties selected from(a) to (f).

In some embodiments, crystalline Compound 2 (Pattern 1) has an X-raypowder diffraction (XRPD) pattern with characteristic peaks at 13.2°2-Theta, 17.2° 2-Theta, 19.3° 2-Theta, 22.4° 2-Theta, and 25.6° 2-Theta.

In some embodiments, crystalline Compound 2 (Pattern 1) has an X-raypowder diffraction (XRPD) pattern substantially the same as shown inFIG. 4.

In some embodiments, crystalline Compound 2 (Pattern 1) has athermo-gravimetric analysis (TGA) or a DSC substantially similar to theones set forth in FIG. 5 and FIG. 6. In some embodiments, crystallineCompound 2 (Pattern 1) has a thermo-gravimetric analysis (TGA)substantially similar to the one set forth in FIG. 5. In someembodiments, crystalline Compound 2 (Pattern 1) has a DSC substantiallysimilar to the one set forth in FIG. 6.

In some embodiments, crystalline Compound 2 (Pattern 1) has an infraredspectrum substantially similar to the one set forth in FIG. 7.

In some embodiments, crystalline Compound 2 (Pattern 1) was obtainedfrom methyl ethyl ketone, acetonitrile, 1,4-dioxane/tert-butyl methylether, methyl ethyl ketone (MEK)/tert-butyl methyl, or ethanol/heptanes.

In some embodiments, Crystalline Pattern 1 of Compound 2 is obtainedfrom:

-   -   (i) methyl ethyl ketone;    -   (ii) methyl ethyl ketone, methyl tert-butyl ether and water;    -   (iii) methyl ethyl ketone, and water;    -   (iv) acetonitrile;    -   (v) 1,4-dioxane and tert-butyl methyl ether;    -   (vi) methyl ethyl ketone and tert-butyl methyl; or    -   (vii) ethanol and heptane.

In one embodiment, Crystalline Pattern 1 of Compound 2 is characterizedby unit cell parameters approximately equal to the following at atemperature of 25° C.:

a(Å) 13.8714(2) b(Å) 7.7379(2) c(Å) 25.5253(5) α ° 90 β° 103.863(1) γ °90 V(Å3) 2659.96(9) Z 4 Calculated Density 1.305 Crystal SystemMonoclinic SG P2₁ R1 0.0301 Sol. Sites 1H₂O

In a further embodiment, Crystalline Pattern 1 of Compound 2 ischaracterized by fractional atomic coordinates substantially the same aslisted in Table 8.

Compound 2—Pattern 2

In some embodiments, Compound 2 is crystalline. In some embodiments,Compound 2 is crystalline Pattern 2. In some embodiments, Compound 2 iscrystalline and has an X-ray powder diffraction (XRPD) patternsubstantially the same as shown in FIG. 8.

Compound 2—Pattern 3

In some embodiments, Compound 2 is crystalline. In some embodiments,Compound 2 is crystalline Pattern 3. In some embodiments, Compound 2 iscrystalline and has an X-ray powder diffraction (XRPD) patternsubstantially the same as shown in FIG. 9.

Prodrugs of Compound 1

In some embodiments, Compound 1 is prepared as a prodrug.

A “prodrug of Compound 1” refers to a compound that is convened intoCompound 1 in vivo. Prodrugs are often useful because, in somesituations, they may be easier to administer than the parent drug. Theymay, for instance, be bioavailable by oral administration whereas theparent is not. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug. In some embodiments,prodrugs facilitate transmittal across a cell membrane where watersolubility is detrimental to mobility but which then is metabolicallyhydrolyzed to the carboxylic acid, the active entity, once inside thecell where water-solubility is beneficial. An example, withoutlimitation, of a prodrug would be an ester of Compound 1 (the“prodrug”). A further example of a prodrug might be a short peptide(polyaminoacid) bonded to an acid group where the peptide is metabolizedto reveal the active moiety.

Prodrugs are generally drug precursors that, following administration toa subject and subsequent absorption, are converted to an active, or amore active species via some process, such as conversion by a metabolicpathway. Some prodrugs have a chemical group present on the prodrug thatrenders it less active and/or confers solubility or some other propertyto the drug. Once the chemical group has been cleaved and/or modifiedfrom the prodrug the active drug is generated. Prodrugs are often usefulbecause, in some situations, they are easier to administer than theparent drug. In certain embodiments, the prodrug of Compound 1 increasesthe bioavailability of Compound 1 when orally administered. In someembodiments, the prodrug of Compound 1 has improved solubility inpharmaceutical compositions over Compound 1.

In some embodiments, a prodrug of Compound 1 is an alkyl ester ofCompound 1, such as, for example, methyl ester, ethyl ester, n-propylester, iso-propyl ester, n-butyl ester, sec-butyl ester, or ten-butylester.

Non-limiting examples of prodrugs of Compound 1 include:

Metabolites of Compound 1

Compound 1 metabolites formed during incubation of Compound 1 with: rat,dog, monkey, and human liver microsomes; rat, dog, and humanhepatocytes; as well as those generated in vivo and isolated from ratbile and rat and dog plasma were investigated. The following metabolitesof Compound 1 were observed:

Metabolite Structure Metabolite Description M1

Glucoronidation of Compound 1 M2

Glucuronidation of Compound 1 plus oxidation M3

Oxidation of phenyl ring of benzyl group. M4

Oxidation of phenyl ring of benzyl group. M5

Oxidation of biphenyl

In some embodiments, sites on Compound 1 are susceptible to variousmetabolic reactions. Therefore incorporation of appropriate substituentson Compound 1 will reduce, minimize or eliminate this metabolic pathway.In specific embodiments, the appropriate substituent to decrease oreliminate the susceptibility of the aromatic ring to metabolic reactionsis, by way of example only, a halogen, deuterium or an alkyl group (e.g.methyl, ethyl).

In some embodiments, Compound 1 is isotopically labeled (e.g. with aradioisotope) or by another other means, including, but not limited to,the use of chromophores or fluorescent moieties, bioluminescent labels,or chemiluminescent labels. In some embodiments, Compound 1 isisotopically-labeled, which is identical to Compound 1 but for the factthat one or more atoms are replaced by an atom having an atomic mass ormass number different from the atomic mass or mass number usually foundin nature. In some embodiments, one or more hydrogen atoms are replacedwith deuterium. In some embodiments, metabolic sites on Compound 1 aredeuterated. In some embodiments, substitution with deuterium affordscertain therapeutic advantages resulting from greater metabolicstability, such as, for example, increased in vivo half-life or reduceddosage requirements.

In one aspect, described is a compound with the following structure:

wherein,

each R is independently selected from hydrogen or deuterium,

or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutically acceptable salt of thecompound is a sodium salt.

Synthesis of Compound 1, and Pharmaceutically Acceptable Salts Thereof

Compound 1, and pharmaceutically acceptable salts thereof (e.g. Compound2), are synthesized as described herein. In additions, solvents,temperatures and other reaction conditions presented herein may vary.

The starting materials used for the synthesis are either synthesized orobtained from commercial sources, such as, but not limited to,Sigma-Aldrich, Fluka, Aeros Organics, Alfa Aesar, VWR Scientific, andthe like. General methods for the preparation of compounds can bemodified by the use of appropriate reagents and conditions for theintroduction of the various moieties found in the structures as providedherein.

In one aspect, the preparation of Compound 1, or pharmaceuticallyacceptable salts thereof (e.g. sodium salt) begins with the stepsoutlined in Scheme 1.

In one aspect, the synthesis of Compound 1 begins with the reaction ofan alkyl acetoacetate with methylamine to provide a compound ofstructure II. Compounds of structure II are reacted with a4-substituted-benzoyl chloride (structure III) to provide compounds ofstructure IV. X is halide, triflate or any other suitable leaving groupfor use in a Suzuki coupling reaction. In some embodiments, X is —Cl,—Br, —I, —OSO₂CF₃, —OSO₂(4-methylphenyl), and —OSO₂CH₃. In someembodiments, X is a halide. In some embodiments, X is —Br. R¹ is analkyl or benzyl. In some embodiments, R² is methyl, ethyl, proproyl, orbenzyl. Other alkyl acetoacetates contemplated include, ethylacetoacetate, isopropyl acetoacetate, benzyl acetoacetate. Treatment ofcompounds of structure IV with hydroxyl amine and acetic acid providesisoxazoles of structure V. Isoxazoles of structure V are utilized asoutlined in scheme 2.

Hydrolysis of the ester group of isoxazoles of structure V providescarboxylic acids of structure VI. Hydrolysis can also be accomplishedwith the use of suitable bases, such as lithium hydroxide or sodiumhydroxide. Suitable solvents for the hydrolysis include water, methanol,ethanol, tetrahydrofuran, or combinations thereof. A Curtiusrearrangement of carboxylic acids of structure VI in the presence of(R)-1-phenylethyl alcohol provides carbamate compounds of structure VII.

In some embodiments, a Suzuki reaction between compounds of structureVII and compounds of structure VIII is used to provide compounds ofstructure X. In some embodiments, R¹ is an alkyl. In some embodiments, Bis boronic acid or boronic ester. In some embodiments, X is

In some embodiments,

In some embodiments, X is —Cl, —Br, —I, —OSO₂CF₃. —OSO₂(4-methylphenyl),and —OSO₂CH₃. In some embodiments, X is a halide. In some embodiments, Xis —Br. In some embodiments, the Suzuki reaction includes the use of apalladium catalyst, a suitable base and a suitable solvent. In someembodiments, the palladium catalyst is a phosphine containing palladiumcatalyst. In some embodiments, the palladium catalyst is Pd(PPh₃)₄ orPd(dppf)Cl₂. In some embodiments, the suitable base for the Suzukireaction is an inorganic base. In some embodiments, the suitable basefor the Suzuki reaction is triethylamine, diisopropylethylamine,1,2,2,6,6-pentamethylpiperidine, tributylamine, sodium bicarbonate,Na₂CO₃, K₂CO₃, Cs₂CO₃, NaOAc, KOAc, Na₃PO₄ or K₃PO₄. Other metalmediated coupling reactions are known for the preparation of compoundsof structure X.

Other metal mediated coupling reactions to form biaryls include, but arenot limited to Suzuki reactions, Stille cross couplings, Negishicouplings, Kumada couplings, Ullmann reactions, Hiyama Coupling, andvariants thereof (Metal-Catalyzed Cross-Coupling Reactions, Armin deMeijere (Editor). François Diederich (Editor), John Wiley & Sons; 2ndedition, 2004; Özdemir, et al., Tetrahedron, 2005, 61, 9791-9798;Ackermann, et al., Org. Lett., 2006, 8, 3457-3460; Blakey, et al. Am.Chem. Soc., 2003, 125, 6046-6047; Dai, et al., Org. Lett., 2004, 6,221-224; Yoshikai, et al, J. Am. Chem. Soc., 2005, 127, 17978-17979;Tang, et al. J. Org. Chem., 2006, 71, 2167-2169; Murata, et al.,Synthesis, 2001, 2231-2233).

In some embodiments, compounds of structure VIII are prepared asoutlined in Scheme 4.

In some embodiments, compounds of structure XI are treated with a dihaloalkyl compound, such as 1,2-dibromoethane, to form a cycloalkyl group.The cyano group is hydrolysed to the acid and an ester is formed fromthe acid to provide tricyclic compounds of structure XII. In someembodiments, compounds of structure XII are reacted with a borylatingagent using transition metal mediated reaction conditions to formboronate compounds of structure VII. In some embodiments, R¹ is ethyl.In some embodiments, X is a halide. In some embodiments, X is —Br.

In some embodiments, compounds of structure VII are reacted with aborylating agent using transition metal mediated reaction conditions toform boronate compounds of structure IX. In some embodiments, theborylating reaction to form IX includes the use of a palladium catalyst,such as Pd(PPh₃)₄ or Pd(dppf)Cl₂, in the presence of a suitable base,such as potassium acetate. In some embodiments, the borylation reagentis selected from among pinacolborane, catecholborane, bis(neopentylglycolato)diboron, bis(pinacolato)diboron, bis(hexyleneglycolato)diboron, and bis(catecholato)diboron. In some embodiments, theborylation reagent is bis(pinacolato)diboron. In some embodiments, theborylation reaction is performed with heating. Boronate compounds ofstructure IX are reacted with compounds of structure XII under palladiummediated coupling conditions (Suzuki reaction conditions) to formcompounds of structure X.

In some embodiments, Compound 1 is prepared as described in Scheme 6.

In some embodiments, biphenyl compounds of structure XIV are elaboratedinto the polycyclic Compound 1 as shown in scheme 6. Biphenyl compoundsof structure XIV are treated with a dihalo alkyl compound, such as1,2-dibromoethane, to form a cycloalkyl group. The cyano group ishydrolysed to the acid and an ester is formed from the acid to providetricyclic compounds of structure XV. In some embodiments, R¹ is ethyl.In some embodiments, R¹ is isopropyl. Tricyclic compounds of structureXV are then treated with acetyl chloride in the presence of a suitableLewis acid, follow by conversion of the acetyl group to the carboxylicacid and treatment of the carboxylic acid with thionyl chloride toprovide acid chlorides of structure XVI. Acid chlorides of structure XVIare then used to prepare isoxazoles of structure XVII as described inScheme 1. In some embodiments, R² is an alkyl group. In someembodiments, R² is methyl and R² is removed from isoxazoles of structureXVII under hydrolysis conditions. In some embodiments, R² is benzyl andR² is removed from isoxazoles of structure XVII under hydrogenationconditions (e.g. H₂, Pd/C). A Curtius rearrangement of carboxylic acidsof structure XVIII in the presence of (R)-1-phenylethyl alcohol providescarbamate compounds of structure X.

An additional alternative route to the synthesis of compound X isoutlined in Scheme 7.

Conversion of the X group in biphenyl compound XX to a boronic acid orboronate ester produces a coupling partner for compound XXI in a Suzukireaction that provides compound X.

The hydrolysis of the ester group in compound X to provide Compound 1and Compound 2 is outlined in Scheme 8.

Hydrolysis of alkyl esters of structure X with a suitable base in asuitable solvent yields Compound 1 after pH adjustment. Suitable basesfor the hydrolysis include, but are not limited to, lithium hydroxideand sodium hydroxide. Suitable solvents for the hydrolysis include, butare not limited to, water, methanol, ethanol, tetrahydrofuran, orcombinations thereof. Compound 1 is then treated with sodium hydroxidein tetrahydrofuran, methanol and water to furnish Compound 2.

In some embodiments, Compound 2 is prepared from compound X byperforming a one-step hydrolysis and salt forming reaction. In someembodiments, the one-step hydrolysis and salt forming reaction includestreatment of compound X with sodium hydroxide in a suitable solvent.

In some embodiments, Compound 1 is treated with potassium hydroxide in asolvent to form Compound 1, potassium salt. In some embodiments,Compound 1 is treated with lithium hydroxide in a solvent to formCompound 1, lithium salt. In some embodiments, Compound 1 is treatedwith calcium hydroxide in a solvent to form Compound 1, calcium salt.

In some embodiments, Compound 1 is treated with dicyclohexylamine in asolvent to form the corresponding salt. In some embodiments, Compound 1is treated with N-methyl-D-glucamine in a solvent to form thecorresponding salt. In some embodiments, Compound 1 is treated withcholine in a solvent to form the corresponding salt. In someembodiments, Compound 1 is treated with tris(hydroxymethyl)methylaminein a solvent to form the corresponding salt.

In some embodiments, Compound 1 is treated with arginine in a solvent toform the corresponding salt. In some embodiments, Compound 1 is treatedwith lysine in a solvent to form the corresponding salt.

In some embodiments, due to the fact that that synthetic methodsdescribed above utilize a transition metal catalyst, purification stepsare performed to reduce the amount of palladium in the product.Purification steps to reduce the amount of palladium in a product areconducted so that active pharmaceutical ingredients meet palladiumspecification guidelines. (“Guideline on the Specification Limits forResidues of Metal Catalysts” European Medicines Agency Pre-authorisationEvaluation of Medicines for Human Use, London, January 2007, Doc. Ref.CPMP/SWP/QWP/4446/00 corr.). In some embodiments, purification steps toreduce the amount of palladium in a product includes, but is not limitedto, treatment with solid trimercaptotriazine (TMT), polystyrene-boundTMT, mercapto-porous polystyrene-bound TMT, polystyrene-boundethylenediamine, activated carbon, glass bead sponges, Smopex™, silicondioxide, silica bound scavengers, thiol-derivatized silica gel,N-acetylcysteine, n-Bu₃P, crystallization, extraction, 1-cysteine,n-Bu₃P/lactic acid. (Garrett et al., Adv. Synth. Catal. 2004, 346,889-900). In some embodiments, activated carbon includes but is notlimited to DARCO® KB-G, DARCO® KB-WJ. In one aspect silica boundscavengers include but are not limited to

where

denotes silica gel. In some embodiments, the purification steps toreduce the amount of palladium include the use of activated carbon,silica gel, derivatized silica gel (e.g. thiol derivatized silica gel),or combinations thereof.

Although the foregoing schemes exemplified the synthesis with(R)-1-phenylethyl alcohol, the same synthetic procedures could beperformed with (S)-1-phenylethyl alcohol or (RS)-1-phenylethyl alcoholin place of (R)-1-phenylethyl alcohol. In some embodiments,(R)-1-phenylethyl alcohol is optically pure. In some embodiments,(R)-1-phenylethyl alcohol has an enantiomeric excess that of at least97%, at least 98%, or at least 99%.

In one aspect, Compound 1 is prepared as outlined in the Examples. Inone aspect, Compound 2 is prepared as outlined in the Examples

Suitable Solvents

Therapeutic agents that are administrable to mammals, such as humans,must be prepared by following regulatory guidelines. Such governmentregulated guidelines are referred to as Good Manufacturing Practice(GMP). GMP guidelines outline acceptable contamination levels of activetherapeutic agents, such as, for example, the amount of residual solventin the final product. Preferred solvents are those that are suitable foruse in GMP facilities and consistent with industrial safety concerns.Categories of solvents are defined in, for example, the InternationalConference on Harmonization of Technical Requirements for Registrationof Pharmaceuticals for Human Use (ICH), “Impurities: Guidelines forResidual Solvents, Q3C(R3), (November 2005).

Solvents are categorized into three classes. Class 1 solvents are toxicand are to be avoided. Class 2 solvents are solvents to be limited inuse during the manufacture of the therapeutic agent. Class 3 solventsare solvents with low toxic potential and of lower risk to human health.Data for Class 3 solvents indicate that they are less toxic in acute orshort-term studies and negative in genotoxicity studies.

Class 1 solvents, which are to be avoided, include: benzene; carbontetrachloride; 1,2-dichloroethane; 1,1-dichloroethene; and1,1,1-trichloroethane.

Examples of Class 2 solvents are: acetonitrile, chlorobenzene,chloroform, cyclohexane, 1,2-dichloroethene, dichloromethane,1,2-dimethoxyethane, N,N-dimethylacetamide, N,N-dimethylformamide,1,4-dioxane, 2-ethoxyethanol, ethyleneglycol, formamide, hexane,methanol, 2-methoxyethanol, methylbutyl ketone, methylcyclohexane,N-methylpyrrolidine, nitromethane, pyridine, sulfolane, tetralin,toluene, 1,1,2-trichloroethene and xylene.

Class 3 solvents, which possess low toxicity, include: acetic acid,acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butylmethylether (MTBE), cumene, dimethyl sulfoxide, ethanol, ethyl acetate, ethylether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropylacetate, methyl acetate, 3-methyl-1-butanol, methylethyl ketone,methylisobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol,1-propanol, 2-propanol, propyl acetate, and tetrahydrofuran.

Residual solvents in active pharmaceutical ingredients (APIs) originatefrom the manufacture of API. In some cases, the solvents are notcompletely removed by practical manufacturing techniques. Appropriateselection of the solvent for the synthesis of APIs may enhance theyield, or determine characteristics such as crystal form, purity, andsolubility. Therefore, the solvent is a critical parameter in thesynthetic process.

In some embodiments, compositions comprising salts of Compound 1comprise an organic solvent(s). In some embodiments, compositionscomprising salts of Compound 1 comprise a residual amount of an organicsolvent(s). In some embodiments, compositions comprising salts ofCompound 1 comprise a residual amount of a Class 3 solvent. In someembodiments, the organic solvent is a Class 3 solvent. In someembodiments, the Class 3 solvent is selected from the group consistingof acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate,tert-butylmethyl ether, cumene, dimethyl sulfoxide, ethanol, ethylacetate, ethyl ether, ethyl formate, formic acid, heptane, isobutylacetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol,methylethyl ketone, methylisobutyl ketone, 2-methyl-1-propanol, pentane,1-pentanol, 1-propanol, 2-propanol, propyl acetate, and tetrahydrofuran.In some embodiments, the Class 3 solvent is selected from ethyl acetate,isopropyl acetate, tert-butylmethylether, heptane, isopropanol, andethanol.

In some embodiments, the compositions comprising a salt of Compound 1include a detectable amount of an organic solvent. In some embodiments,the salt of Compound 1 is a sodium salt (i.e. Compound 2). In someembodiments, the organic solvent is a Class 3 solvent.

In one aspect, the salt of Compound 1 is a sodium salt, potassium salt,lithium salt, calcium salt, magnesium salt, ammonium salt, choline salt,protonated dicyclohexylamine salt, protonated N-methyl-D-glucamine salt,protonated tris(hydroxymethyl)methylamine salt, arginine salt, or lysinesalt. In one aspect, the salt of Compound 1 is a sodium salt.

In other embodiments are compositions comprising Compound 2, wherein thecomposition comprises a detectable amount of solvent that is less thanabout 1%, wherein the solvent is selected from acetone,1,2-dimethoxyethane, acetonitrile, ethyl acetate, tetrahydrofuran,methanol, ethanol, heptane, and 2-propanol. In a further embodiment arecompositions comprising Compound 2, wherein the composition comprises adetectable amount of solvent which is less than about 5000 ppm. In yet afurther embodiment are compositions comprising Compound 2, wherein thedetectable amount of solvent is less than about 5000 ppm, less thanabout 4000 ppm, less than about 3000 ppm, less than about 2000 ppm, lessthan about 1000 ppm, less than about 500 ppm, or less than about 100ppm.

Certain Terms

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Unlessotherwise indicated, conventional methods of mass spectroscopy, NMR,HPLC, protein chemistry, organic synthesis, biochemistry, recombinantDNA techniques and pharmacology, within the skill of the art areemployed. In this application, the use of“or” means “and/or” unlessstated otherwise. Furthermore, use of the term “including” as well asother forms, such as “include”, “includes,” and “included,” is notlimiting.

The term “pharmaceutically acceptable excipient,” as used herein, refersto a material, such as a carrier, diluent, stabilizer, dispersing agent,suspending agent, thickening agent, etc. which allows processing theactive pharmaceutical ingredient (API) into a form suitable foradministration to a mammal. In one aspect, the mammal is a human.Pharmaceutically acceptable excipients refer to materials which do notsubstantially abrogate the desired biological activity or desiredproperties of the compound (i.e. API), and is relatively nontoxic, i.e.,the material is administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

“Active pharmaceutical ingredient” or API refers to a compound thatpossesses a desired biological activity or desired properties. In someembodiments, an API is Compound 1. In some embodiments, an API isCompound 2. Provided herein is an active pharmaceutical ingredient(API), Compound 1, or pharmaceutically acceptable salt thereof (e.g.Compound 2), with a purity of greater than 80%, greater than 85%,greater than 90%, greater than 95%, greater than 96%, greater than 97%,greater than 98%, greater than 98%, or greater than 99%. In specificembodiments, provided herein is an active pharmaceutical ingredient(API), Compound 2, with a purity of greater than 80%, greater than 85%,greater than 90%, greater than 95%, greater than 96%, greater than 97%,greater than 98%, or greater than 99%.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients. e.g. Compound 1 or a pharmaceutically acceptable salt, anda co-agent, are both administered to a patient simultaneously in theform of a single entity or dosage. The term “non-fixed combination”means that the active ingredients, e.g. Compound 1 or a pharmaceuticallyacceptable salt, and a co-agent, are administered to a patient asseparate entities either simultaneously, concurrently or sequentiallywith no specific intervening time limits, wherein such administrationprovides effective levels of the two compounds in the body of thepatient. The latter also applies to cocktail therapy, e.g. theadministration of three or more active ingredients.

The term “pharmaceutical composition” refers to a mixture of Compound 1,or pharmaceutically acceptable salt and/or solvate thereof, with otherchemical components, such as carriers, stabilizers, diluents, dispersingagents, suspending agents, thickening agents, excipients, etc. Thepharmaceutical composition facilitates administration of the compound toa mammal.

Administration of a combination of agents, as used herein, includesadministration of the agents described in a single composition or in acombination therapy wherein one or more agent is administered separatelyfrom at least one other agent.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylmoiety is branched, straight chain, or cyclic. The alkyl group may bedesignated as “C₁-C₆alkyl”. In one aspect, an alkyl is selected frommethyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl,neopentyl, hexyl, ethenyl, propenyl, allyl, butenyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and the like.

“Detectable amount” refers to an amount that is measurable usingstandard analytic methods (e.g. ion chromatography, mass spectrometry,NMR, HPLC, gas chromatography, elemental analysis, IR spectroscopy,inductively coupled plasma atomic emission spectrometry, USP<231>MethodII, etc) (ICH guidances, Q2A Test on Validation of Analytical Procedures(March 1995) and Q2 Validation of Analytical Procedures: Methodology(November 1996)).

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of an agent being administeredwhich will relieve to some extent one or more of the symptoms of thedisease or condition being treated. The result can be reduction and/oralleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. For example, an “effectiveamount” for therapeutic uses is the amount of the composition comprisinga compound as disclosed herein required to provide a clinicallysignificant decrease in disease symptoms. The term “therapeuticallyeffective amount” includes, for example, a prophylactically effectiveamount. The effective amount will be selected based on the particularpatient and the disease level. It is understood that “an effect amount”or “a therapeutically effective amount” varies from subject to subject,due to variation in metabolism of drug, age, weight, general conditionof the subject, the condition being treated, the severity of thecondition being treated, and the judgment of the prescribing physician.In one embodiment, an appropriate “effective” amount in any individualcase is determined using techniques, such as a dose escalation study

The terms “co-administration” or the like, as used herein, are meant toencompass administration of the selected therapeutic agents to a singlepatient, and are intended to include treatment regimens in which theagents are administered by the same or different route of administrationor at the same or different time.

The terms “enhance” or “enhancing,” as used herein, means to increase orprolong either in potency or duration a desired effect. Thus, in regardto enhancing the effect of therapeutic agents, the term “enhancing”refers to the ability to increase or prolong, either in potency orduration, the effect of other therapeutic agents on a system. An“enhancing-effective amount,” as used herein, refers to an amountadequate to enhance the effect of another therapeutic agent in a desiredsystem.

The terms “kit” and “article of manufacture” are used as synonyms.

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized(biotransformed). The term “metabolized.” as used herein, refers to thesum of the processes (including, but not limited to, hydrolysisreactions and reactions catalyzed by enzymes) by which a particularsubstance is changed by an organism. Thus, enzymes may produce specificstructural alterations to a compound. For example, cytochrome P450catalyzes a variety of oxidative and reductive reactions while uridinediphosphate glucuronyltransferases (UGT) catalyze the transfer of anactivated glucuronic-acid molecule to aromatic alcohols, aliphaticalcohols, carboxylic acids, amines and free sulphydryl groups (e.g.conjugation reactions). In some embodiments, compounds disclosed hereinare metabolite to provide taurine metabolites. Further information onmetabolism is available in The Pharmacological Basis of Therapeutics,9th Edition, McGraw-Hill (1996). In one embodiment, metabolites of thecompounds disclosed herein are identified either by administration ofcompounds to a host and analysis of tissue samples from the host, or byincubation of compounds with hepatic cells in vitro and analysis of theresulting compounds.

The term “modulate,” as used herein, means to interact with a targeteither directly or indirectly so as to alter the activity of the target,including, by way of example only, to enhance the activity of thetarget, to inhibit the activity of the target, to limit the activity ofthe target, or to extend the activity of the target.

The term “modulator,” as used herein, refers to a molecule thatinteracts with a target either directly or indirectly. The interactionsinclude, but are not limited to, the interactions of an agonist and anantagonist.

The term “agonist,” as used herein, refers to a molecule such as acompound, a drug, an enzyme activator or a hormone modulator that bindsto a specific receptor and triggers a response in the cell. An agonistmimics the action of an endogenous ligand (such as prostaglandin,hormone or neurotransmitter) that binds to the same receptor.

The term “antagonist,” as used herein, refers to a molecule such as acompound, which diminishes, inhibits, or prevents the action of anothermolecule or the activity of a receptor site.

The term “LPA-dependent”, as used herein, refers to conditions ordisorders that would not occur, or would not occur to the same extent,in the absence of LPA.

The term “LPA-mediated”, as used herein, refers to refers to conditionsor disorders that might occur in the absence of LPA but can occur in thepresence of LPA.

The term “subject” or “patient” encompasses mammals. In one aspect, themammal is a human.

In another aspect, the mammal is a non-human primate such as chimpanzee,and other apes and monkey species. In one aspect, the mammal is a farmanimal such as cattle, horse, sheep, goat, or swine. In one aspect, themammal is a domestic animal such as rabbit, dog, or cat. In one aspect,the mammal is a laboratory animal, including rodents, such as rats, miceand guinea pigs, and the like.

“Bioavailability” refers to the percentage of the weight of Compound 1,or a pharmaceutically acceptable salt and/or solvate thereof, dosed thatis delivered into the general circulation of the animal or human beingstudied. The total exposure (AUC_((0-∞))) of a drug when administeredintravenously is usually defined as 100% Bioavailable (F %). “Oralbioavailability” refers to the extent to which Compound 1, or apharmaceutically acceptable salt and/or solvate thereof, is absorbedinto the general circulation when the pharmaceutical composition istaken orally as compared to intravenous injection.

“Blood plasma concentration” refers to the concentration Compound 1, inthe plasma component of blood of a mammal. It is understood that theplasma concentration of Compound 1 may vary significantly betweensubjects, due to variability with respect to metabolism and/orinteractions with other therapeutic agents. In one aspect, the bloodplasma concentration of Compound 1 varies from subject to subject.Likewise, values such as maximum plasma concentration (C_(max)), or timeto reach maximum plasma concentration (T_(max)), or total area under theplasma concentration time curve (AUC_((0-∞))) vary from subject tosubject. Due to this variability, in one embodiment, the amountnecessary to constitute “a therapeutically effective amount” of Compound1 varies from subject to subject.

“Drug absorption” or “absorption” typically refers to the process ofmovement of drug from site of administration of a drug across a barrierinto a blood vessel or the site of action, e.g., a drug moving from thegastrointestinal tract into the portal vein or lymphatic system.

“Serum concentration” or “Plasma concentration” describes the bloodserum or blood plasma concentration, typically measured in mg, μg, or ngof therapeutic agent per ml, dl, or 1 of blood serum, absorbed into thebloodstream after administration. Plasma concentrations are typicallymeasured in ng/ml or μg/ml.

“Pharmacodynamics” refers to the factors which determine the biologicresponse observed relative to the concentration of drug at a site ofaction.

“Pharmacokinetics” refers to the factors which determine the attainmentand maintenance of the appropriate concentration of drug at a site ofaction.

“Steady state,” as used herein, is when the amount of drug administeredis equal to the amount of drug eliminated within one dosing intervalresulting in a plateau or constant plasma drug exposure.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating at least one symptom of a diseaseor condition, preventing additional symptoms, inhibiting the disease orcondition, e.g., arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

Pharmaceutical Compositions/Formulations

Pharmaceutical compositions are formulated in a conventional mannerusing one or more physiologically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which are used pharmaceutically. Suitabletechniques, carriers, and excipients include those found within, forexample, Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., PharmaceuticalDosage Forms, Marcel Decker, New York, N.Y., 1980; and PharmaceuticalDosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams& Wilkins 1999), herein incorporated by reference in their entirety.

A pharmaceutical composition, as used herein, refers to a mixture of acompound of Formula (I) with other chemical components (i.e.pharmaceutically acceptable inactive ingredients), such as carriers,excipients, binders, filling agents, suspending agents, flavoringagents, sweetening agents, disintegrating agents, dispersing agents,surfactants, lubricants, colorants, diluents, solubilizers, moisteningagents, plasticizers, stabilizers, penetration enhancers, wettingagents, anti-foaming agents, antioxidants, preservatives, or one or morecombination thereof. The pharmaceutical composition facilitatesadministration of the compound to an organism.

Pharmaceutical formulations described herein are administerable to asubject in a variety of ways by multiple administration routes,including but not limited to, oral, parenteral (e.g., intravenous,subcutaneous, intramuscular, intramedullary injections, intrathecal,direct intraventricular, intraperitoneal, intralymphatic, intranasalinjections), intranasal, buccal, topical or transdermal administrationroutes. The pharmaceutical formulations described herein include, butare not limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations, and mixed immediateand controlled release formulations.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is administered orally.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is administered topically. In suchembodiments, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, shampoos, scrubs, rubs, smears, medicated sticks,medicated bandages, balms, creams or ointments. In one aspect, Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2), isadministered topically to the skin.

In another aspect, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is administered by inhalation.

In another aspect, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is formulated for intranasal administration.Such formulations include nasal sprays, nasal mists, and the like.

In another aspect, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is formulated as eye drops.

In any of the aforementioned aspects are further embodiments in whichthe effective amount of Compound 1, or a pharmaceutically acceptablesalt thereof (e.g. Compound 2), is: (a) systemically administered to themammal; and/or (b) administered orally to the mammal; and/or (c)intravenously administered to the mammal; and/or (d) administered byinhalation to the mammal; and/or (e) administered by nasaladministration to the mammal; or and/or (f) administered by injection tothe mammal; and/or (g) administered topically to the mammal; and/or (h)administered by ophthalmic administration; and/or (i) administeredrectally to the mammal; and/or (j) administered non-systemically orlocally to the mammal.

In any of the aforementioned aspects are further embodiments comprisingsingle administrations of the effective amount of the compound,including further embodiments in which (i) the compound is administeredonce; (ii) the compound is administered to the mammal multiple timesover the span of one day; (iii) continually; or (iv) continuously.

In any of the aforementioned aspects are further embodiments comprisingmultiple administrations of the effective amount of the compound,including further embodiments in which (i) the compound is administeredcontinuously or intermittently; as in a single dose; (ii) the timebetween multiple administrations is every 6 hours; (iii) the compound isadministered to the mammal every 8 hours; (iv) the compound isadministered to the mammal every 12 hours; (v) the compound isadministered to the mammal every 24 hours. In further or alternativeembodiments, the method comprises a drug holiday, wherein theadministration of the compound is temporarily suspended or the dose ofthe compound being administered is temporarily reduced; at the end ofthe drug holiday, dosing of the compound is resumed. In one embodiment,the length of the drug holiday varies from 2 days to 1 year.

In certain embodiments, a compound as described herein is administeredin a local rather than systemic manner.

In some embodiments, the compound described herein is administeredtopically. In some embodiments, the compound described herein isadministered systemically.

In some embodiments, for oral administration, Compound 1, or apharmaceutically acceptably salt thereof (e.g. Compound 2), areformulated by combining the active compound with pharmaceuticallyacceptable carriers or excipients. Such carriers enable Compound 1, or apharmaceutically acceptably salt thereof (e.g. Compound 2) to beformulated as tablets, powders, pills, dragees, capsules, liquids, gels,syrups, elixirs, slurries, suspensions and the like, for oral ingestionby a patient to be treated. In some embodiments, for oraladministration, Compound 1, or a pharmaceutically acceptably saltthereof (e.g. Compound 2), is formulated without combining the activecompound with pharmaceutically acceptable carriers or excipients and isplaced directly into a capsule for administration to a mammal.

In some embodiments, the pharmaceutical compositions will include atleast one pharmaceutically acceptable carrier, diluent or excipient andCompound 1 as an active ingredient in free-acid or free-base form, or ina pharmaceutically acceptable salt form. In some embodiments, thepharmaceutical compositions will include at least one pharmaceuticallyacceptable carrier, diluent or excipient and Compound 2.

The pharmaceutical compositions described herein include Compound 1, ora pharmaceutically acceptable salt thereof (e.g. Compound 2). In someembodiments, the pharmaceutical compositions described herein includeCompound 1. In some embodiments, the pharmaceutical compositionsdescribed herein include amorphous Compound 1. In some embodiments, thepharmaceutical compositions described herein include crystallineCompound 1. In some embodiments, the pharmaceutical compositionsdescribed herein include Compound 2. In some embodiments, thepharmaceutical compositions described herein include amorphous Compound2. In some embodiments, the pharmaceutical compositions described hereininclude crystalline Compound 2.

In some embodiments, the pharmaceutical compositions described hereininclude: (a) Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2); and one or more of the following: (b) binders; (c)disintegrants; (d) fillers (diluents); (e) lubricants; (f) glidants(flow enhancers); (g) compression aids; (h) colors; (i) sweeteners; (j)preservatives; (k) suspensing/dispersing agents; (l) filmformers/coatings; (m) flavors; (o) printing inks; (p) solubilizers; (q)alkalizing agents; (r) buffering agents; (s) antioxidants; (t)effervsescent agents.

In some embodiments, the pharmaceutical compositions described hereininclude: (a) Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2); and (b) a capsule shell.

In some embodiments, pharmaceutical compositions described hereininclude one or more of the following in addition to Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2): (a)magnesium stearate; (b) lactose; (c) microcrystalline cellulose; (d)silicified microcrystalline cellulose; (e) mannitol; (f) starch (corn);(g) silicon dioxide; (h) titanium dioxide; (i) stearic acid; (j) sodiumstarch glycolate; (k) gelatin; (l) talc; (m) sucrose; (n) aspartame; (o)calcium stearate; (p) povidone; (q) pregelatinized starch; (r) hydroxypropyl methylcellulose; (s) OPA products (coatings & inks); (t)croscarmellose; (u) hydroxy propyl cellulose; (v) ethylcellulose; (w)calcium phosphate (dibasic); (x) crospovidone; (y) shellac (and glaze);(z) sodium carbonate; (aa) hypromellose.

In one embodiment, pharmaceutical preparations for oral use are obtainedby mixing one or more solid excipient with one or more of the compoundsdescribed herein, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as: for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methylcellulose, microcrystalline cellulose, silicified microcrystallinecellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose;or others such as: polyvinylpyrrolidone (PVP or povidone) or calciumphosphate. If desired, disintegrating agents are added, such as thecross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, oralginic acid or a salt thereof such as sodium alginate.

In one embodiment, the pharmaceutical compositions described herein areformulated into any suitable dosage form, including but not limited to,aqueous oral dispersions, solid oral dosage forms, fast meltformulations, effervescent formulations, lyophilized formulations,tablets, capsules, pills, controlled release formulations, entericcoated tablets, inhaled powder, inhaled dispersion, IV formulations.

In further embodiments, the pharmaceutical compositions provided hereinmay be provided as compressed tablets, tablet triturates, rapidlydissolving tablets, multiple compressed tablets, or enteric-coatedtablets, sugar-coated, or film-coated tablets.

Pharmaceutical dosage forms can be formulated in a variety of methodsand can provide a variety of drug release profiles, including immediaterelease, sustained release, and delayed release. In some cases it may bedesirable to prevent drug release after drug administration until acertain amount of time has passed (i.e. timed release), to providesubstantially continuous release over a predetermined time period (i.e.sustained release) or to provide release immediately following drugadministration (i.e., immediate release).

In some embodiments, formulations provide a therapeutically effectiveamount of Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), enabling, for example, once a week, twice a week,three times a week, four times a week, five times a week, once everyother day, once-a-day, twice-a-day (b.i.d.), or three times a day(t.i.d.) administration if desired. In one embodiment, the formulationprovides a therapeutically effective amount of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) enablingonce-a-day administration.

In one embodiment, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is formulated into an immediate release formthat provides for once-a-day administration. Generally speaking, onewill desire to administer an amount of Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2) that is effective to achieve aplasma level commensurate with the concentrations found to be effectivein vivo for a period of time effective to elicit a therapeutic effect.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) and one or more excipients are dry blended andcompressed into a mass, such as a tablet, having a hardness sufficientto provide a pharmaceutical composition that substantially disintegrateswithin less than about 10 minutes, less than about 15 minutes, less thanabout 20 minutes, less than about 25 minutes, less than about 30minutes, less than about 35 minutes, or less than about 40 minutes,after oral administration, thereby releasing the Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) formulationinto the gastrointestinal fluid.

In some embodiments, the pharmaceutical compositions provided herein inan immediate release dosage form are capable of releasing not less than75% of the therapeutically active ingredient or combination and/or meetthe disintegration or dissolution requirements for immediate releasetablets of the particular therapeutic agents or combination included inthe tablet core, as set forth in USP XXII, 1990 (The United StatesPharmacopeia.). Immediate release pharmaceutical compositions includecapsules, tablets, pills, oral solutions, powders, beads, pellets,particles, and the like.

Excipients used in pharmaceutical compositions should be selected on thebasis of compatibility with Compound 1, or a pharmaceutically acceptablesalt thereof (e.g. Compound 2) and the release profile properties of thedesired dosage form. Exemplary excipients include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike.

Binders impart cohesiveness to solid oral dosage form formulations: forpowder filled capsule formulation, they aid in plug formation that isfilled into soft or hard shell capsules and for tablet formulation, theyensure the tablet remaining intact after compression and help assureblend uniformity prior to a compression or fill step.

In some embodiments, the binder(s) are selected from starches, sugars,povidone, cellulose or modified cellulose such as microcrystallinecellulose, hydroxypropyl methyl cellulose, lactose, or sugar alcoholslike xylitol, sorbitol or maltitol. In some embodiments, the binder ishydroxypropyl methyl cellulose. In some embodiments, the binder ishypromellose (e.g., Methocel E5).

In general, binder levels of 20-70% are used in powder-filled gelatincapsule formulations. Binder usage level in tablet formulations varieswhether direct compression, wet granulation, roller compaction, or usageof other excipients such as fillers which itself acts as moderatebinder.

Dispersing agents, and/or viscosity modulating agents include materialsthat control the diffusion and homogeneity of a drug through liquidmedia or a granulation method or blend method. In some embodiments,these agents also facilitate the effectiveness of a coating or erodingmatrix.

Diluents increase bulk of the composition to facilitate compression orcreate sufficient bulk for homogenous blend for capsule filling.

The term “disintegrate” includes both the dissolution and dispersion ofthe dosage form when contacted with gastrointestinal fluid.“Disintegration agents or disintegrants” facilitate the breakup ordisintegration of a substance. In some embodiments, one aspect, solidoral dosage forms include up to 15% w/w of disintegrant. In someembodiments, the disintegrant is croscarmellose sodium. In anotheraspect, the disintegrant is sodium starch glycolate or crospovidone.

Filling agents include compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

In one aspect, the filler is lactose (e.g. monohydrate). In anotheraspect, the filler is mannitol, or dicalcium phosphate. In anotheraspect, the filler is mannitol, microcrystalline cellulose, dicalciumphosphate or sorbitol.

Gastrointestinal fluid is the fluid of stomach secretions of a subjector the saliva of a subject after oral administration of a compositiondescribed herein, or the equivalent thereof. An “equivalent of stomachsecretion” includes, e.g., an in vitro fluid having similar contentand/or pH as stomach secretions such as a 1% sodium dodecyl sulfatesolution or 0.1N HCl solution in water. In addition, simulatedintestinal fluid (USP) is an aqueous phosphate buffer system at pH 6.8.

Lubricants and glidants are compounds that prevent, reduce or inhibitadhesion or friction of materials. In one aspect, solid oral dosageforms include about 0.25% w/w to about 2.5% w/w of lubricant. In anotheraspect solid oral dosage forms include about 0.5% w/w to about 1.5% w/wof lubricant.

In some embodiments, the solid dosage forms described herein are in theform of a tablet, (including an immediate release tablet, an extendedrelease tablet, a sustained release tablet, a enteric coated tablet, asuspension tablet, a fast-melt tablet, a bite-disintegration tablet, arapid-disintegration tablet, an effervescent tablet, or a caplet), apill, a powder (including a sterile packaged powder, a dispensablepowder, or an effervescent powder), a capsule (including both soft orhard capsules, e.g., capsules made from animal-derived gelatin orplant-derived HPMC, or “sprinkle capsules”), solid dispersion,multiparticulate dosage forms, pellets, or granules.

In other embodiments, the pharmaceutical formulation is in the form of apowder. In still other embodiments, the pharmaceutical formulation is inthe form of a tablet, including but not limited to, an immediate releasetablet. Additionally, pharmaceutical formulations described herein areadministered as a single dosage or in multiple dosages. In someembodiments, the pharmaceutical formulation is administered in two, orthree, or four tablets.

In some embodiments, solid dosage forms, e.g., tablets, effervescenttablets, and capsules, are prepared by mixing Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) with one ormore pharmaceutical excipients to form a bulk blend composition. Whenreferring to these bulk blend compositions as homogeneous, it is meantthat the Compound 1, or a pharmaceutically acceptable salt thereof (e.g.Compound 2) particles are dispersed evenly throughout the composition sothat the composition is capable of being readily subdivided into equallyeffective unit dosage forms, such as tablets, pills, or capsules. In oneembodiment, the individual unit dosages also include film coatings,which disintegrate upon oral ingestion or upon contact with diluent. Inone embodiment, these formulations are manufactured by conventionaltechniques.

Conventional techniques include, e.g., one or a combination of methods:(1) dry mixing, (2) direct compression, (3) milling, (4) dry ornon-aqueous granulation, (5) wet granulation, or (6) fusion. See, e.g.,Lachman et al., The Theory and Practice of Industrial Pharmacy (1986).Other methods include, e.g., spray drying, pan coating, meltgranulation, granulation, fluidized bed spray drying or coating (e.g.,wurster coating), tangential coating, top spraying, tableting, extrudingand the like.

Compressed tablets are solid dosage forms prepared by compacting thebulk blend formulations described above. In various embodiments,compressed tablets which are designed to dissolve in the mouth willinclude one or more flavoring agents. In other embodiments, thecompressed tablets will include a film surrounding the final compressedtablet. In some embodiments, the film coating aids in patient compliance(e.g., Opadry® coatings or sugar coating). Film coatings comprisingOpadry® typically range from about 1% to about 5% of the tablet weight.In other embodiments, the compressed tablets include one or moreexcipients.

Provided herein are pharmaceutical compositions in film-coated dosageforms, which comprise a combination of an active ingredient, or apharmaceutically acceptable salt, solvate, or prodrug thereof; and oneor more tabletting excipients to form a tablet core using conventionaltabletting processes and subsequently coating the core. The tablet corescan be produced using conventional granulation methods, for example wetor dry granulation, with optional commination of the granules and withsubsequent compression and coating.

Further provided herein are pharmaceutical compositions in entericcoated dosage forms, which comprise a combination of an activeingredient, or a pharmaceutically acceptable salt, solvate, or prodrugthereof; and one or more release controlling excipients for use in anenteric coated dosage form. The pharmaceutical compositions alsocomprise non-release controlling excipients.

Enteric-coatings are coatings that resist the action of stomach acid butdissolve or disintegrate in the intestine.

In one aspect, the oral solid dosage form disclosed herein include anenteric coating(s). Enteric coatings include one or more of thefollowing: cellulose acetate phthalate; methyl acrylate-methacrylic acidcopolymers; cellulose acetate succinate; hydroxy propyl methyl cellulosephthalate; hydroxy propyl methyl cellulose acetate succinate(hypromellose acetate succinate); polyvinyl acetate phthalate (PVAP);methyl methacrylate-methacrylic acid copolymers; methacrylic acidcopolymers, cellulose acetate (and its succinate and phthalate version);styrol maleic acid co-polymers; polymethacrylic acid/acrylic acidcopolymer; hydroxyethyl ethyl cellulose phthalate; hydroxypropyl methylcellulose acetate succinate; cellulose acetate tetrahydrophtalate;acrylic resin; shellac.

An enteric coating is a coating put on a tablet, pill, capsule, pellet,bead, granule, particle, etc. so that it doesn't dissolve until itreaches the small intestine.

Sugar-coated tablets are compressed tablets surrounded by a sugarcoating, which may be beneficial in covering up objectionable tastes orodors and in protecting the tablets from oxidation.

Film-coated tablets are compressed tablets that are covered with a thinlayer or film of a water-soluble material. Film coatings include, butare not limited to, hydroxyethylcellulose, sodiumcarboxymethylcellulose, polyethylene glycol 4000, and cellulose acetatephthalate. Film coating imparts the same general characteristics assugar coating. Multiple compressed tablets are compressed tablets madeby more than one compression cycle, including layered tablets, andpress-coated or dry-coated tablets. In some embodiments, tablets arecoated with water soluble, pH independent film coating which allows forimmediate disintegration for fast, active release (e.g. Opadryproducts).

In some embodiments, the pharmaceutical compositions provided herein arein the form of a controlled release dosage form. As used herein, theterm “controlled release” refers to a dosage form in which the rate orplace of release of the active ingredient(s) is different from that ofan immediate dosage form when orally administered. Controlled releasedosage forms include delayed-, extended-, prolonged-, sustained-,pulsatile-, modified-, targeted-, programmed-release. The pharmaceuticalcompositions in controlled release dosage forms are prepared using avariety of modified release devices and methods including, but notlimited to, matrix controlled release devices, osmotic controlledrelease devices, multiparticulate controlled release devices,ion-exchange resins, enteric coatings, multilayered coatings, andcombinations thereof. The release rate of the active ingredient(s) canalso be modified by varying the particle sizes.

In contrast to immediate release compositions, controlled releasecompositions allow delivery of an agent to a human over an extendedperiod of time according to a predetermined profile. Such release ratescan provide therapeutically effective levels of agent for an extendedperiod of time and thereby provide a longer period of pharmacologicresponse. Such longer periods of response provide for many inherentbenefits that are not achieved with the corresponding immediate releasepreparations. In one aspect, controlled release compositions of Compound1, or a pharmaceutically acceptable salt thereof, providetherapeutically effective levels of Compound 1 for an extended period oftime and thereby provide a longer period of pharmacologic response.

Delayed release as used herein refers to the delivery so that therelease can be accomplished at some generally predictable location inthe intestinal tract more distal to that which would have beenaccomplished if there had been no delayed release alterations. In someembodiments the method for delay of release is coating. Any coatingsshould be applied to a sufficient thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below about 5,but does dissolve at pH about 5 and above.

In some embodiments, the pharmaceutical compositions provided herein isin a modified release dosage form that is fabricated using a matrixcontrolled release device (see. Takada et al in “Encyclopedia ofControlled Drug Delivery,” Vol. 2, Mathiowitz ed., Wiley, 1999).

In one embodiment, the pharmaceutical compositions provided herein in amodified release dosage form is formulated using an erodible matrixdevice, which is water-swellable, erodible, or soluble polymers,including synthetic polymers, and naturally occurring polymers andderivatives, such as polysaccharides and proteins.

In some embodiments, a matrix controlled release system includes anenteric coating so that no drug is released in the stomach.

The pharmaceutical compositions provided herein may be provided inunit-dosage forms or multiple-dosage forms. Unit-dosage forms, as usedherein, refer to physically discrete units suitable for administrationto human and animal subjects and packaged individually as is known inthe art. Each unit-dose contains a predetermined quantity of the activeingredient(s) sufficient to produce the desired therapeutic effect, inassociation with the required pharmaceutical carriers or excipients.Examples of unit-dosage forms include individually packaged tablets andcapsules. Unit-dosage forms may be administered in fractions ormultiples thereof. A multiple-dosage form is a plurality of identicalunit-dosage forms packaged in a single container to be administered insegregated unit-dosage form. Examples of multiple-dosage forms includebottles of tablets or capsules.

In other embodiments a powder comprising the Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) formulationsdescribed herein are formulated to include one or more pharmaceuticalexcipients and flavors. Additional embodiments also comprise asuspending agent and/or a wetting agent. This bulk blend is uniformlysubdivided into unit dosage packaging or multi-dosage packaging units.The term “uniform” means the homogeneity of the bulk blend issubstantially maintained during the packaging process.

In still other embodiments, effervescent powders are prepared.Effervescent salts have been used to disperse medicines in water fororal administration. Effervescent salts are granules or coarse powderscontaining a medicinal agent in a dry mixture, usually composed ofsodium bicarbonate, citric acid and/or tartaric acid.

The method of preparation of the effervescent granules described hereinemploys three basic processes: wet granulation, dry granulation andfusion. The fusion method is used for the preparation of most commercialeffervescent powders. It should be noted that, although these methodsare intended for the preparation of granules, the formulations ofeffervescent salts described herein, in one embodiment, are alsoprepared as tablets, according to technology for tablet preparation.

In one embodiment, pharmaceutical preparations which are used orallyinclude push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a plasticizer, such as glycerol orsorbitol. In one embodiment, the push-fit capsules contain the activeingredients in admixture with filler such as lactose, binders such asstarches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In one embodiment, the push-fit capsulescontain the active ingredient only without additional inactiveingredients. In one embodiment, in soft capsules, the active compoundsare dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition, in oneembodiment, stabilizers are added. In other embodiments, the formulationis placed in a sprinkle capsule, wherein the capsule is swallowed wholeor the capsule is opened and the contents sprinkled on food prior toeating.

All formulations for oral administration should be in dosages suitablefor such administration.

In some embodiments, pharmaceutical formulations are provided comprisingCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2) and at least one dispersing agent or suspending agent for oraladministration to a subject. In one embodiment, the formulation is apowder and/or granules for suspension, and upon admixture with water, asubstantially uniform suspension is obtained.

A suspension is “substantially uniform” when it is mostly homogenous,that is, when the suspension is composed of approximately the sameconcentration of Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) at any point throughout the suspension (USPChapter 905).

Liquid formulation dosage forms for oral administration are aqueoussuspensions or non-aqueous suspensions.

Liquid formulation dosage forms for oral administration are aqueoussuspensions selected from, but not limited to, pharmaceuticallyacceptable aqueous oral dispersions, emulsions, solutions, and syrups.See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology,2^(nd) Ed., pp. 754-757 (2002). In addition to including Compound 1, ora pharmaceutically acceptable salt thereof (e.g. Compound 2), the liquiddosage forms include additives, such as: (a) disintegrating agents; (b)dispersing agents; (c) wetting agents; (d) preservatives; (c) viscosityenhancing agents; (f) sweetening agents; (g) flavoring agents; (h)solibizing agents (bioavailability enhancers).

In one embodiment, the aqueous suspensions and dispersions describedherein remain in a homogenous state, as defined above by USP Chapter905, for at least 4 hours.

Liquid compositions illustratively take the form of a liquid where theagent (e.g. Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2)) is present in solution, in suspension or both. In oneembodiment, the liquid composition is aqueous.

Liquid compositions illustratively take the form of a liquid where theagent (e.g. Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2)) is present in solution, in suspension or both. In oneembodiment, the liquid composition is non-aqueous.

In one embodiment, the aqueous suspension also contains one or morepolymers as suspending agents. Useful polymers include water-solublepolymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. In one embodiment, useful compositionsalso comprise an mucoadhesive polymer, selected for example fromcarboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

In one embodiment, pharmaceutical compositions also include one or morepH adjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium carbonate,sodium citrate, sodium acetate, sodium lactate andtris-hydroxymethylaminomethane; and buffers such as citrate/dextrose,sodium carbonate, sodium bicarbonate and ammonium chloride. Such acids,bases and buffers are included in an amount required to maintain pH ofthe composition in an acceptable range.

In one embodiment, liquid pharmaceutical compositions also include oneor more salts in an amount required to bring osmolality of thecomposition into an acceptable range. Such salts include those havingsodium, potassium or ammonium cations and chloride, citrate, ascorbate,borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfiteanions; suitable salts include sodium chloride, potassium chloride,sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In one embodiment, pharmaceutical compositions also include one or morepreservatives to inhibit microbial activity.

Still other compositions include one or more surfactants to enhancephysical stability or for other purposes. Suitable nonionic surfactantsinclude polyoxyethylene fatty acid glycerides and vegetable oils, e.g.,polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylenealkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.

Still other compositions include one or more antioxidants to enhancechemical stability where required. Suitable antioxidants include, by wayof example only, ascorbic acid, tocopherol, and sodium metabisulfite.

In one embodiment, aqueous compositions are packaged in single-dosenon-reclosable containers. Alternatively, multiple-dose reclosablecontainers are used, in which case it is typical to include apreservative in the composition.

In some embodiments, aqueous pharmaceutical compositions do not includea preservative and are used within 24 hours of preparation.

In some embodiments, aqueous pharmaceutical compositions include one ormore solubilizers which aid in enhancing the bioavailability of theactive pharmaceutical ingredient. In some embodiments, the solubilizeris selected from Labrasol, Lutrol (macrogels, poloxamers), and othersknown in the art.

The oral pharmaceutical solutions described herein are beneficial forthe administration to infants (less than 2 years old), children under 10years of age and any patient group that is unable to swallow or ingestsolid oral dosage forms.

For buccal or sublingual administration, in one embodiment, thecompositions take the form of tablets, lozenges, or gels formulated in aconventional manner (see e.g. U.S. Pat. Nos. 4,229,447, 4,596,795,4,755,386, and 5,739,136).

In one embodiment, dragee cores are prepared with suitable coatings. Forthis purpose, concentrated sugar solutions are used, which optionallycontain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. In one embodiment,dyestuffs or pigments are added to the tablets or dragee coatings foridentification or to characterize different combinations of activecompound doses.

It should be understood that many carriers and excipients may serveseveral functions, even within the same formulation.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is formulated in the form of a pharmaceuticalcomposition that is suitable for inhalation/nasal delivery. In someembodiments, the pharmaceutical composition is in the form of asolution, suspension, emulsion, colloidal dispersion, spray, dry powder,aerosol, or combinations thereof. In some embodiments, thepharmaceutical composition comprises at least one pharmaceuticallyacceptable excipient that is commonly used in nasal/inhalablepharmaceutical compositions. In some embodiments, the pharmaceuticalcomposition is administered with an atomizer, an insufflator, anebulizer, a vaporizer, or a metered dose inhaler. In some embodiments,the pharmaceutical composition is inhaled nasally or orally. In someembodiments, crystalline Compound 1 is used in the pharmaceuticalcomposition. In some embodiments, crystalline Compound 2 is used in thepharmaceutical composition. In some embodiments, amorphous Compound 1 isused in the pharmaceutical composition. In some embodiments, amorphousCompound 2 is used in the pharmaceutical composition.

Representative nasal/inhalation formulations are described in, forexample, Ansel, H. C. et al., Pharmaceutical Dosage Forms and DrugDelivery Systems, Sixth Ed. (1995); REMINGTON: THE SCIENCE AND PRACTICEOF PHARMACY, 21st edition, 2005.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is formulated in the form of a nasal spray,nasal mist, and the like.

For administration by inhalation, Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2) is formulated for use as anaerosol, a mist or a powder.

In some embodiments, pharmaceutical compositions suitable fornasal/inhalation administration are conveniently delivered in the formof an aerosol spray presentation from pressurized packs or a nebuliser,with the use of a suitable propellant. Capsules and cartridges for usein an inhaler or insufflator may be formulated containing a powder mixof the compound described herein and a suitable powder base such aslactose or starch.

In some embodiments, the pharmaceutical composition is in the form of apowder for nasal/inhalation delivery to a mammal. In some embodiments,powders comprise micronized and/or nano-sized particles of Compound 1,or a pharmaceutically acceptable salt thereof (e.g. Compound 2), blendedwith larger carrier particles that prevent aggregation. For example, inone embodiment a dry powder formulation is prepared as follows: Compound1 or a pharmaceutically acceptable salt thereof (e.g. Compound 2) is jetmilled. Lactose is jet milled and the two ingredients are mixed and thefinal mixture is packaged in sterile insufflators. In some instancespowder inhalable formulations described herein comprise crystallineparticles of Compound 1. In some instances powder inhalable formulationsdescribed herein comprise crystalline particles of Compound 2. In someembodiments, powder inhalable formulations described herein compriseamorphous particles of Compound 1. In some embodiments, powder inhalableformulations described herein comprise amorphous particles of Compound2.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is prepared as transdermal dosage forms. Inone embodiment, the transdermal formulations described herein include atleast three components: (1) a formulation of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2); (2) apenetration enhancer; and (3) an aqucous adjuvant. In some embodimentsthe transdermal formulations include additional components such as, butnot limited to, gelling agents, creams and ointment bases, and the like.In some embodiments, the transdermal formulation further include a wovenor non-woven backing material to enhance absorption and prevent theremoval of the transdermal formulation from the skin. In otherembodiments, the transdermal formulations described herein can maintaina saturated or supersaturated state to promote diffusion into the skin.

In one aspect, formulations suitable for transdermal administration ofcompounds described herein employ transdermal delivery devices andtransdermal delivery patches and can be lipophilic emulsions orbuffered, aqueous solutions, dissolved and/or dispersed in a polymer oran adhesive. In one aspect, such patches are constructed for continuous,pulsatile, or on demand delivery of pharmaceutical agents. Stillfurther, transdermal delivery of the compounds described herein can beaccomplished by means of iontophoretic patches and the like. In oneaspect, transdermal patches provide controlled delivery of the activecompound. In one aspect, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound to the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2), is formulated into a pharmaceutical compositionsuitable for intramuscular, subcutaneous, or intravenous injection. Inone aspect, formulations suitable for intramuscular, subcutaneous, orintravenous injection include physiologically acceptable sterile aqueousor non-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.In some embodiments, formulations suitable for subcutaneous injectionalso contain additives such as preserving, wetting, emulsifying, anddispensing agents. In some cases it is desirable to include isotonicagents, such as sugars, sodium chloride, and the like. Prolongedabsorption of the injectable pharmaceutical form can be brought about bythe use of agents delaying absorption, such as aluminum monostearate andgelatin.

For intravenous injections, compounds described herein are formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. For other parenteral injections, appropriateformulations include aqueous or nonaqueous solutions, preferably withphysiologically compatible buffers or excipients. Such excipients areknown.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. In one aspect, the active ingredient is in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is administered topically and can beformulated into a variety of topically administrable compositions, suchas solutions, suspensions, lotions, gels, pastes, medicated sticks,balms, creams or ointments. Such pharmaceutical compounds can containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2), is administered topically and can beformulated into a variety of topically administrable compositions, suchas solutions, suspensions, lotions, gels, pastes, medicated sticks,balms, creams or ointments.

Dose Amounts of Compound 1 or a Pharmaceutically Acceptable Salt Thereof(e.g. Compound 2)

In certain embodiments, the effective amount of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) is about 1 mgto about 2 g per dose, about 1 mg to about 1.5 g per dose, about 5 mg toabout 1500 mg per dose or about 10 mg to about 1500 mg per dose. In someembodiments, the effective amount of Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2) is about 1 mg to about 5 g perday, about 5 mg to about 2 g per day, about 5 mg to about 1.5 g per day,about 10 mg to about 1.5 g per day, or about 10 mg to about 1 g per day.

In one embodiment, the effective amount of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) is about 5 mgper dose, about 10 mg per dose, about 15 mg per dose, about 20 mg perdose, about 25 mg per dose, about 50 mg per dose, about 100 mg per dose,about 150 mg per dose, about 200 mg per dose, about 250 mg per dose,about 300 mg per dose, about 350 mg per dose, about 400 mg per dose,about 450 mg per dose, about 500 mg per dose, about 550 mg per dose,about 600 mg per dose, about 650 mg per dose, about 700 mg per dose,about 750 mg per dose, about 800 mg per dose, about 850 mg per dose,about 900 mg per dose, about 1000 mg per dose or about 1500 mg per dose.

In some embodiments, oral pharmaceutical solutions include about 0.0.01mg/ml to about 10 mg/ml of Compound 2. In some embodiments, oralpharmaceutical solutions include about 1 mg/ml to about 10 mg/ml ofCompound 2.

In one aspect, immediate release tablets include about 5% w/w to about50% w/w of Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2). In some embodiments, immediate release tabletsinclude about 5% w/w to about 40% w/w, or about 5% w/w to about 30% w/wof Compound 1, or a pharmaceutically acceptable salt thereof (e.g.Compound 2). In some embodiments, immediate release tablets includeabout 5% w/w, about 10% w/w, about 15% w/w, about 20% w/w, about 25%w/w, about 30% w/w, about 33% w/w, about 35% w/w, about 40% w/w ofCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2).

In one aspect, immediate release capsules include about 1.25% w/w toabout 50% w/w of Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2). In some embodiments, immediate releasecapsules include Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) and the capsule shell only.

Methods of Dosing and Treatment Regimens

In one embodiment, the pharmaceutical compositions including Compound 1,or a pharmaceutically acceptable salt thereof (e.g. Compound 2),described herein is administered for prophylactic and/or therapeutictreatments. In therapeutic applications, the compositions areadministered to a patient already suffering from a disease or condition,in an amount sufficient to cure or at least partially arrest at leastone of the symptoms of the disease or condition. In certain embodiments,amounts effective for this use depend on the severity and course of thedisease or condition, previous therapy, the patient's health status,weight, and response to the drugs, and/or the judgment of the treatingphysician.

In prophylactic applications, compositions containing Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), describedherein are administered to a patient susceptible to or otherwise at riskof a particular disease, disorder or condition. Such an amount isdefined to be a “prophylactically effective amount or dose.” In thisuse, the precise amounts also depend on the patient's state of health,weight, and the like. When used in a patient, effective amounts for thisuse will depend on the severity and course of the disease, disorder orcondition, previous therapy, the patient's health status and response tothe drugs, and the judgment of the treating physician.

In certain embodiments, administration of the compound, compositions ortherapies as described herein includes chronic administration. Incertain embodiments, chronic administration includes administration foran extended period of time, including, e.g., throughout the duration ofthe patient's life in order to ameliorate or otherwise control or limitthe symptoms of the patient's disease or condition. In some embodiments,chronic administration includes daily administration.

In some embodiments, administration of the compounds, compositions ortherapies described herein is given continuously. In alternativeembodiments, the dose of drug being administered is temporarily reducedor temporarily suspended for a certain length of time (i.e., a “drugholiday”). The length of the drug holiday varies between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250days, 280 days, 300 days, 320 days, 350 days, and 365 days. The dosereduction during a drug holiday is from 10%-100%, including by way ofexample only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, and 100%.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is administered once a day to a mammal in needthereof. In some embodiments, Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2) is administered twice a day toa mammal in need thereof. In some embodiments, Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) isadministered three times a day to a mammal in need thereof.

In some embodiments, the compounds, compositions or therapies describedherein are administered in at least one priming dose, followed by atleast one maintenance dose. In certain embodiments, a priming dose ofthe agent(s) is administered until the symptoms of the disorder, diseaseor condition treated have been reduced (e.g., to a satisfactory level).Upon reduction, a maintenance dose of the compounds, compositions ortherapies described herein is administered if desired or if necessary.In some embodiments, the maintenance dose comprises administration ofthe agent(s) described herein in an amount sufficient to at leastpartially maintain the reduction achieved by administration of thepriming dose. In various embodiments, the maintenance dose, compared tothe priming dose, includes a decrease in dosage and/or frequency ofadministration of the agent or one or more of the agents administered inthe method. In certain embodiments, however, intermittent treatment withincreased frequency and/or dosage amounts may be necessary upon anyrecurrence of symptoms.

In certain embodiments, the amount of a given agent that corresponds toa priming or maintenance amount varies depending upon factors including,by way of non-limiting example, the specific agent(s) utilized, thedisease condition and its severity, the identity (e.g., weight) of thesubject or host in need of treatment, and/or the route ofadministration. In various embodiments, the desired dose is convenientlypresented in a single dose or in divided doses administeredsimultaneously (or over a short period of time) or at appropriateintervals, for example as two, three, four or more sub-doses per day.

Pharmacokinetic and Pharmacodynamic Analysis

In one embodiment, any standard pharmacokinetic protocol is used todetermine blood plasma concentration profile in humans followingadministration of a formulation described herein (that include Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2)). Forexample, a randomized single-dose crossover study is performed using agroup of healthy adult human subjects. The number of subjects issufficient to provide adequate control of variation in a statisticalanalysis, and is typically about 10 or greater, although for certainpurposes a smaller group suffices. Each subject receives administrationat time zero a single dose of a formulation of Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), normally ataround 8 am following an overnight fast. The subjects continue to fastand remain in an upright position for about 2 hours after administrationof the formulation. Blood samples are collected from each subject priorto administration (e.g., 15 minutes) and at several intervals afteradministration. In certain instances, several samples are taken withinthe first hour and taken less frequently thereafter. Illustratively,blood samples are collected at 0 (pre-dose), 0.25, 0.5, 1, 2, 3, 4, 6,8, 12, and 16 hours after administration and, 24, 36, 48, 60 and 72hours after administration. If the same subjects are to be used forstudy of a second test formulation, a period of at least 10 days shouldelapse before administration of the second formulation. Plasma isseparated from the blood samples by centrifugation and the separatedplasma is analyzed for Compound 1 by a validated high performance liquidchromatography/tandem weight spectrometry (LC/APCI-MS/MS) procedure suchas, for example, Ramu et al., Journal of Chromatography B, 751 (2001)49-59).

Any formulation giving the desired pharmacokinetic profile is suitablefor administration according to the present methods.

Patient Selection

In any of the aforementioned aspects involving the prevention ortreatment of LPA-mediated diseases or conditions are further embodimentscomprising identifying patients by screening for LPA receptor gene SNPs.A SNP located in the promoter region of LPA₁ showed significantassociation with knee osteoarthritis in two independent populations(Mototani et al. Hum. Mol. Genetics, vol. 17, no. 12, 2008). Patientscan be further selected based on increased LPA receptor expression inthe tissue of interest. For example, chronic lymphocytic leukemia (CLL)is characterized by the accumulation of CD19+/CD5+ B-lymphocytes in theperipheral blood, bone marrow and lymphoid organs which occurs as aresult of a block in B-lymphocyte apoptosis. LPA can protect some CLLcells from apoptosis and the cells that are protected by LPA have highlevels of LPA₁ mRNA. In some embodiments, CLL patients are selectedbased on the expression of the LPA1R. LPA receptor expression aredetermined by methods including, but not limited to, northern blotting,western blotting, quantitative PCR (qPCR), flow cytometry,autoradiography (using a small molecule radioligand or PET ligand). Insome embodiments, patients are selected based on the concentration ofserum or tissue LPA measured by mass spectrometry. LPA concentrationsare high in ovarian cancer ascites and in some breast cancer effusions.In some embodiments, patients are selected based on a combination of theabove markers (increased LPA concentrations and increased LPA receptorexpression).

Combination Therapies

In certain instances, it is appropriate to administer Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) incombination with another therapeutic agent. By way of example only, ifone of the side effects experienced by a patient upon receiving one ofthe compounds herein is inflammation, then it may be appropriate toadminister an anti-inflammatory agent in combination with the initialtherapeutic agent.

Or, in one embodiment, the therapeutic effectiveness of one of thecompounds described herein is enhanced by administration of an adjuvant(i.e., by itself the adjuvant may have minimal therapeutic benefit, butin combination with another therapeutic agent, the overall therapeuticbenefit to the patient is enhanced). Or, in some embodiments, thebenefit experienced by a patient is increased by administering one ofthe compounds described herein with another therapeutic agent (whichalso includes a therapeutic regimen) that also has therapeutic benefit.

In one specific embodiment, Compound 1, or a pharmaceutically acceptablesalt thereof (e.g. Compound 2) is co-administered with a secondtherapeutic agent, wherein Compound 1, or a pharmaceutically acceptablesalt thereof (e.g. Compound 2) and the second therapeutic agent modulatedifferent aspects of the disease, disorder or condition being treated,thereby providing a greater overall benefit than administration ofeither therapeutic agent alone.

In any case, regardless of the disease, disorder or condition beingtreated, the overall benefit experienced by the patient may simply beadditive of the two therapeutic agents or the patient may experience asynergistic benefit.

In certain embodiments, different therapeutically-effective dosages ofthe compounds disclosed herein will be utilized in formulatingpharmaceutical composition and/or in treatment regimens when thecompounds disclosed herein are administered in combination with one ormore additional agent, such as an additional therapeutically effectivedrug, an adjuvant or the like. Therapeutically-effective dosages ofdrugs and other agents for use in combination treatment regimens can bedetermined by means similar to those set forth hereinabove for theactives themselves. Furthermore, the methods of prevention/treatmentdescribed herein encompasses the use of metronomic dosing, i.e.,providing more frequent, lower doses in order to minimize toxic sideeffects. In some embodiments, a combination treatment regimenencompasses treatment regimens in which administration of Compound 1, ora pharmaceutically acceptable salt thereof (e.g. Compound 2) isinitiated prior to, during, or after treatment with a second agentdescribed herein, and continues until any time during treatment with thesecond agent or after termination of treatment with the second agent. Italso includes treatments in which Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2) and the second agent beingused in combination are administered simultaneously or at differenttimes and/or at decreasing or increasing intervals during the treatmentperiod. Combination treatment further includes periodic treatments thatstart and stop at various times to assist with the clinical managementof the patient.

Compositions and methods for combination therapy are provided herein. Inaccordance with one aspect, the pharmaceutical compositions disclosedherein are used to treat LPA-dependent or LPA-mediated conditions.

It is understood that the dosage regimen to treat, prevent, orameliorate the condition(s) for which relief is sought, is modified inaccordance with a variety of factors. These factors include the disease,disorder or condition from which the subject suffers, as well as theage, weight, sex, diet, and medical condition of the subject. Thus, insome instances, the dosage regimen actually employed varies and, in someembodiments, deviates from the dosage regimens set forth herein.

For combination therapies described herein, dosages of theco-administered compounds vary depending on the type of co-drugemployed, on the specific drug employed, on the disease or conditionbeing treated and so forth. In additional embodiments, whenco-administered with one or more other therapeutic agents, the compoundprovided herein is administered either simultaneously with the one ormore other therapeutic agents, or sequentially.

In combination therapies, the multiple therapeutic agents (one of whichis one of the compounds described herein) are administered in any orderor even simultaneously. If administration is simultaneous, the multipletherapeutic agents are, by way of example only, provided in a single,unified form, or in multiple forms (e.g., as a single pill or as twoseparate pills). In one embodiment, one of the therapeutic agents isgiven in multiple doses, and in another, two (or more if present) aregiven as multiple doses. In some embodiments of non-simultaneousadministration, the timing between the multiple doses vary from morethan zero weeks to less than four weeks. In addition, the combinationmethods, compositions and formulations are not to be limited to the useof only two agents; the use of multiple therapeutic combinations is alsoenvisioned.

Compound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2) and combination therapies are administered before, during or afterthe occurrence of a disease or condition, and the timing ofadministering the composition containing a compound varies. Thus, in oneembodiment, the compounds described herein are used as a prophylacticand are administered continuously to subjects with a propensity todevelop conditions or diseases in order to prevent the occurrence of thedisease or condition. In another embodiment, the compounds andcompositions are administered to a subject during or as soon as possibleafter the onset of the symptoms. In specific embodiments, a compounddescribed herein is administered as soon as is practicable after theonset of a disease or condition is detected or suspected, and for alength of time necessary for the treatment of the disease. In someembodiments, the length required for treatment varies, and the treatmentlength is adjusted to suit the specific needs of each subject. Forexample, in specific embodiments, a compound described herein or aformulation containing the compound is administered for at least 2weeks, about 1 month to about 5 years.

By way of example, therapies which combine Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) withinhibitors of LPA synthesis or LPA receptor antagonists, either actingat the same or other points in the LPA synthesis or signalling pathway,are encompassed herein for treating LPA-dependent or LPA-mediateddiseases or conditions.

In another embodiment described herein, methods for treatment ofLPA-dependent or LPA-mediated conditions or diseases, such asproliferative disorders, including cancer, comprises administration to amammal Compound 1, or a pharmaceutically acceptable salt thereof (e.g.Compound 2) in combination with at least one additional agent selected,by way of example only, alemtuzumab, arsenic trioxide, asparaginase(pegylated or non-), bevacizumab, cetuximab, platinum-based compoundssuch as cisplatin, cladribine, daunorubicin/doxorubicin/idarubicin,irinotecan, fludarabine, 5-fluorouracil, gemtuzumab, methotrexate,Paclitaxel™, taxol, temozolomide, thioguanine, or classes of drugsincluding hormones (an antiestrogen, an antiandrogen, or gonadotropinreleasing hormone analogues, interferons such as alpha interferon,nitrogen mustards such as busulfan or melphalan or mechlorethamine,retinoids such as tretinoin, topoisomerase inhibitors such as irinotecanor topotecan, tyrosine kinase inhibitors such as gefinitinib orimatinib, or agents to treat signs or symptoms induced by such therapyincluding allopurinol, filgrastim, granisetron/ondansetron/palonosetron,dronabinol.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2) is administered or formulated in combination with oneor more anti-cancer agents. In some embodiments, one or more of theanti-cancer agents are proapoptotic agents. Examples of anti-canceragents include, but are not limited to, any of the following: gossypol,genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA),bryostatin, tumor necrosis factor-related apoptosis-inducing ligand(TRAIL), 5-aza-2′-deoxycytidine, all trans retinoic acid, doxorubicin,vincristine, etoposide, gemcitabine, imatinib, geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, or PD184352,Taxol™ (paclitaxel), and analogs of Taxol™, such as Taxotere™. Compoundsthat have the basic taxane skeleton as a common structure feature, havealso been shown to have the ability to arrest cells in the G2-M phasesdue to stabilized microtubules and may be useful for treating cancer incombination with the compounds described herein.

Further examples of anti-cancer agents for use in combination withCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2) include inhibitors of mitogen-activated protein kinase signaling,e.g., U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063,SP600125, BAY 43-9006, wortmannin, or LY294002; Syk inhibitors; mTORinhibitors; and antibodies (e.g., rituxan).

Other anti-cancer agents for use in combination with Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) include oneor more of the following: abiraterorone; abarelix; adriamycin;aactinomycin; acivicin; aclarubicin; acodazole hydrochloride; acronine;adozelesin; aldesleukin; alemtuzumab; allopurinol; alitretinoin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;aminolevulinic acid; amifostine; amsacrine; anastrozole; anthramycin;aprepitant; arsenic trioxide; asparaginase; asperlin; azacitidine;azetepa; azotomycin; batimastat; bendamustine hydrochloride; benzodepa;bevacizumab; bexarotene; bicalutamide; bisantrene hydrochloride;bisnafide dimesylate; bizelesin; bleomycin; bleomycin sulfate;bortezomib; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; capecitabine; cedefingol; cetuximab;chlorambucil; cirolemycin; cisplatin; cladribine; clofarabine; crisnatolmesylate; cyclophosphamide; cytarabine; dacarbazine; dasatinib;daunorubicin hydrochloride; dactinomycin; darbepoetin alfa; decitabine;degarelix; denileukin diftitox; dexormaplatin; dexrazoxanehydrochloride; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; eltrombopag olamine; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; epoetin alfa; erbulozole;erlotinib hydrochloride; esorubicin hydrochloride; estramustine;estramustine phosphate sodium; etanidazole; etoposide; etoposidephosphate; etoprine; everolimus; exemestane; fadrozole hydrochloride;fazarabine; fenretinide; filgrastim; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; fulvestrant;gefitinib; gemcitabine; gemcitabine hydrochloride;gemcitabine-cisplatin; gemtuzumab ozogamicin; goserelin acetate;histrelin acetate; hydroxyurea; idarubicin hydrochloride; ifosfamide;iimofosine; ibritumomab tiuxetan; idarubicin; ifosfamide; imatinibmesylate; imiquimod; interleukin II (including recombinant interleukinII, or rlL2), interferon alfa-2a; interferon alfa-2b; interferonalfa-n1; interferon alfa-n3; interferon beta-1a; interferon gamma-1b;iproplatin; irinotecan hydrochloride; ixabepilone; lanreotide acetate;lapatinib; lenalidomide; letrozole; leuprolide acetate; leucovorincalcium; leuprolide acetate; levamisole; liposomal cytarabine; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; methoxsalen; metoprine; meturedepa;mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycinC; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid;nandrolone phenpropionate; nelarabine; nilotinib; nocodazoic;nofetumomab; nogalamycin; ofatumumab; oprelvekin; ormaplatin;oxaliplatin; oxisuran; paclitaxel; palifermin; palonosetronhydrochloride; pamidronate; pegfilgrastim; pemetrexed disodium;pentostatin; panitumumab; pazopanib hydrochloride; pemetrexed disodium;plerixafor; pralatrexate; pegaspargase; peliomycin; pentamustine;peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantronehydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin;prednimustine; procarbazine hydrochloride; puromycin; puromycinhydrochloride; pyrazofurin; quinacrine; raloxifene hydrochloride;rasburicase; recombinant HPV bivalent vaccine; recombinant HPVquadrivalent vaccine; riboprine; rogletimide; rituximab; romidepsin;romiplostim; safingol; safingol hydrochloride; sargramostim; semustine;simtrazene; sipuleucel-T; sorafenib; sparfosate sodium; sparsomycin;spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin;streptozocin; sulofenur; sunitinib malate; talisomycin; tamoxifencitrate; tecogalan sodium; tegafur, teloxantrone hydrochloride;temozolomide; temoporfin; temsirolimus; teniposide; teroxirone;testolactone; thalidomide; thiamiprine; thioguanine; thiotepa;tiazofurin; tirapazamine; topotecan hydrochloride; toremifene;tositumomab; tositumomab and I 131 Iodine tositumomab; trastuzumab;trestolone acetate; tretinoin; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; valrubicin; vapreotide; verteporfin; vinblastine;vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate;vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate;vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate;vorinostat; vorozole; zeniplatin; zinostatin; zoledronic acid; zorubicinhydrochloride.

Yet other anticancer agents for use in combination include alkylatingagents, antimetabolites, natural products, or hormones, e.g., nitrogenmustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, etc.),alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine,lomusitne, etc.), or triazenes (decarbazine, etc.). Examples ofantimetabolites include but are not limited to folic acid analog (e.g.,methotrexate), or pyrimidine analogs (e.g., Cytarabine), purine analogs(e.g., mercaptopurine, thioguanine, pentostatin).

Examples of natural products for use in combination with Compound 1, ora pharmaceutically acceptable salt thereof (e.g. Compound 2) include butare not limited to vinca alkaloids (e.g., vinblastin, vincristine),epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin,doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), or biologicalresponse modifiers (e.g., interferon alpha).

Examples of alkylating agents for use in combination with Compound 1, ora pharmaceutically acceptable salt thereof (e.g. Compound 2) include,but are not limited to, nitrogen mustards (e.g., mechloroethamine,cyclophosphamide, chlorambucil, meiphalan, etc.), ethylenimine andmethylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates(e.g., busulfan), nitrosourcas (e.g., carmustine, lomusitne, semustine,streptozocin, etc.), or triazenes (decarbazine, etc.). Examples ofantimetabolites include, but are not limited to folic acid analog (e.g.,methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine,Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine,pentostatin.

Examples of hormones and antagonists for use in combination withCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2) include, but are not limited to, adrenocorticosteroids (e.g.,prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrolacetate, medroxyprogesterone acetate), estrogens (e.g.,diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen),androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen(e.g., flutamide), gonadotropin releasing hormone analog (e.g.,leuprolide). Other agents that can be used in the methods andcompositions described herein for the treatment or prevention of cancerinclude platinum coordination complexes (e.g., cisplatin, carboblatin),anthracenedione (e.g., mitoxantrone), substituted urea (e.g.,hydroxyurca), methyl hydrazine derivative (e.g., procarbazine),adrenocortical suppressant (e.g., mitotane, aminoglutethimide).

Examples of anti-cancer agents which act by arresting cells in the G2-Mphases due to stabilized microtubules include without limitation thefollowing marketed drugs and drugs in development: Erbulozole,Dolastatin 10, Mivobulin isethionate, Vincristine, NSC-639829,Discodermolide, ABT-751, Altorhyrtins (such as Altorhyrtin A andAltorhyrtin C), Spongistatins (such as Spongistatin 1, Spongistatin 2,Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6,Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotinhydrochloride, Epothilones (such as Epothilone A, Epothilone B,Epothilone C, Epothilone D, Epothilone E, Epothilone F, Epothilone BN-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothiloneB, 21-hydroxyepothilone D, 26-fluoroepothilone, Auristatin PE,Soblidotin, Vincristine sulfate, Cryptophycin 52, Vitilevuamide,Tubulysin A, Canadensol, Centaureidin, Oncocidin A1 Fijianolide B,Laulimalide, Narcosine, Nascapine, Hemiasterlin, Vanadoceneacetylacetonate, Indanocine Eleutherobins (such asDesmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A, andZ-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, DiazonamideA, Taccalonolide A, Diozostatin, (−)-Phenylahistin, Myoseverin B,Resverastatin phosphate sodium.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2) is co-administered with thrombolytic agents (e.g.,alteplase anistreplase, streptokinase, urokinase, or tissue plasminogenactivator), heparin, tinzaparin, warfarin, dabigatran (e.g., dabigatranetexilate), factor Xa inhibitors (e.g., fondaparinux, draparinux,rivaroxaban, DX-9065a, otamixaban, LY517717, or YM150), ticlopidine,clopidogrel, CS-747 (prasugrel, LY640315), ximelagatran, or BIBR 1048.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is used in combination with anti-emetic agentsto treat nausea or emesis. Anti-emetic agents include, but are notlimited to: neurokinin-1 receptor antagonists, 5HT3 receptor antagonists(such as ondansetron, granisetron, tropisetron, Palonosetron, andzatisetron), GABA_(B) receptor agonists (such as baclofen),corticosteroids (such as dexamethasone, prednisone, prednisolone, orothers), dopamine antagonists (such as, but not limited to, domperidone,droperidol, haloperidol, chlorpromazine, promethazine, prochlorperazine,metoclopramide), antihistamines (H1 histamine receptor antagonists, suchas but not limited to, cyclizine, diphenhydramine, dimenhydrinate,meclizine, promethazine, hydroxyzine), cannabinoids (such as but notlimited to, cannabis, marinol, dronabinol), and others (such as, but notlimited to, trimethobenzamide; ginger, emetrol, propofol).

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is used in combination with an agent useful inthe treatment of anemia. Such an anemia treatment agent is, for example,a continuous eythropoiesis receptor activator (such as epoetin-α).

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is used in combination with an agent useful inthe treatment of neutropenia. Examples of agents useful in the treatmentof neutropenia include, but are not limited to, a hematopoietic growthfactor which regulates the production and function of neutrophils suchas a human granulocyte colony stimulating factor, (G-CSF). Examples of aG-CSF include filgrastim.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is used in combination with radiation therapy(or radiotherapy). Radiation therapy is the treatment of cancer andother diseases with ionizing radiation. Radiation therapy can be used totreat localized solid tumors, such as cancers of the skin, tongue,larynx, brain, breast, prostate, colon, uterus and/or cervix. It canalso be used to treat leukemia and lymphoma (cancers of theblood-forming cells and lymphatic system, respectively).

A technique for delivering radiation to cancer cells is to placeradioactive implants directly in a tumor or body cavity. This is calledinternal radiotherapy (brachytherapy, interstitial irradiation, andintracavitary irradiation are types of internal radiotherapy.) Usinginternal radiotherapy, the radiation dose is concentrated in a smallarea, and the patient stays in the hospital for a few days. Internalradiotherapy is frequently used for cancers of the tongue, uterus,prostate, colon, and cervix.

The term “radiotherapy” or “ionizing radiation” include all forms ofradiation, including but not limited to α, β, and γ radiation andultraviolet light.

In one aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2) is used to treat or reduce fibrosis in a mammal. Inone aspect, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2) is administered in combination with one or moreimmunosuppressants. Immunosuppressive therapy is clinically used totreat or prevent the rejection of transplanted organs and tissues (e.g.bone marrow, heart, kidney, liver); treatment of autoimmune diseases ordiseases that are most likely of autoimmune origin (e.g. rheumatoidarthritis, myasthenia gravis, systemic lupus erythematosus, Crohn'sdisease, and ulcerative colitis); and treatment of some othernon-autoimmune inflammatory diseases (e.g. long term allergic asthmacontrol), and in the treatment of fibrotic conditions.

In some embodiments, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is administered with corticosteroids. In someembodiments, Compound 1, or a pharmaceutically acceptable salt thereof(e.g. Compound 2) is administered with an a therapeutic agent selectedfrom among: Calcineurin inhibitors (such as, but not limited to,cyclosporin, tacrolimus); mTOR inhibitors (such as, but not limited to,sirolimus, everolimus); anti-proliferatives (such as, but not limitedto, azathioprine, mycophenolic acid); corticosteroids (such as, but notlimited to, prednisone, cortisone acetate, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone acetate, deoxycorticosterone acetate,aldosterone, hydrocortisone); antibodies (such as, but not limited to,monoclonal anti-IL-2Rα receptor antibodies (basiliximab, daclizumab),polyclonal anti-T-cell antibodies (anti-thymocyte globulin (ATG),anti-lymphocyte globulin (ALG)), B-cell antagonists, rituximab,natalizumab.

Other therapeutic agents include, but are not limited to:cyclophosphamide, penicillamine, cyclosporine, nitrosoureas, cisplatin,carboplatin, oxaliplatin, methotrexate, azathioprine, mercaptopurine,pyrimidine analogues, protein synthesis inhibitors, dactinomycin,anthracyclines, mitomycin C, bleomycin, mithramycin, Atgam®,Thymoglobuline®, OKT3®, basiliximab, daclizumab, cyclosporin,tacrolimus, sirolimus, Interferons (IFN-β, IFN-γ), opioids, TNF bindingproteins (infliximab, etanercept, adalimumab, golimumab), leflunomide,gold thioglucose, gold thiomalate, aurofin, sulfasalazine,hydroxychloroquinine, minocycline, rapamicin, mycophenolic acid,mycophenolate mofetil, FTY720, as well as those listed in U.S. Pat. No.7,060,697.

In one embodiment, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is administered in combination withCyclosporin A (CsA) or tacrolimus (FK506). In one embodiment, Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2) isadministered to a mammal in combination with an anti-inflammatory agentincluding, but not limited to, non-steroidal anti-inflammatory drugs(NSAIDs) and corticosteroids (glucocorticoids).

NSAIDs include, but are not limited to: aspirin, salicylic acid,gentisic acid, choline magnesium salicylate, choline salicylate, cholinemagnesium salicylate, choline salicylate, magnesium salicylate, sodiumsalicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium,flurobiprofen, ibuprofen, ketoprofen, nabutone, ketolorac, ketorolactromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin,sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid,piroxicam, meloxicam, COX-2 specific inhibitors (such as, but notlimited to, celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib,lumiracoxib, CS-502, JTE-522, L-745,337 and NS398).

Corticosteroids, include, but are not limited to: betamethasone,prednisone, alclometasone, aldosterone, amcinonide, beclometasone,betamethasone, budesonide, ciclesonide, clobetasol, clobetasone,clocortolone, cloprednol, cortisone, cortivazol, deflazacort,deoxycorticosterone, desonide, desoximetasone, desoxycortone,dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone,fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone,fluperolone, fluprednidene, fluticasone, formocortal, halcinonide,halometasone, hydrocortisone/cortisol, hydrocortisone aceponate,hydrocortisone buteprate, hydrocortisone butyrate, loteprednol,medrysone, meprednisone, methylprednisolone, methylprednisoloneaceponate, mometasone furoate, paramethasone, prednicarbate,prednisone/prednisolone, rimexolone, tixocortol, triamcinolone, andulobetasol.

In one embodiment, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is administered in combination withleukotriene receptor antagonists.

In another embodiment described herein, methods for treatment ofLPA-dependent or LPA-mediated conditions or diseases, such asatherosclerosis, comprises administration to a patient compounds,pharmaceutical compositions, or medicaments described herein incombination with at least one additional agent selected, by way ofexample only, HMG-CoA reductase inhibitors (e.g., statins in theirlactonized or dihydroxy open acid forms and pharmaceutically acceptablesalts and esters thereof, including but not limited to lovastatin;simvastatin; dihydroxy open-acid simvastatin, particularly the ammoniumor calcium salts thereof; pravastatin, particularly the sodium saltthereof; fluvastatin, particularly the sodium salt thereof;atorvastatin, particularly the calcium salt thereof; nisvastatin, alsoreferred to as NK-104; rosuvastatin); agents that have bothlipid-altering effects and other pharmaceutical activities; HMG-CoAsynthase inhibitors; cholesterol absorption inhibitors such asezetimibe; cholesterol ester transfer protein (CETP) inhibitors, forexample JTT-705 and CP529, 414; squalene epoxidase inhibitors; squalenesynthetase inhibitors (also known as squalene synthase inhibitors);acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors includingselective inhibitors of ACAT-1 or ACAT-2 as well as dual inhibitors ofACAT-1 and -2; microsomal triglyceride transfer protein (MTP)inhibitors; probucol; niacin; bile acid sequestrants; LDL (low densitylipoprotein) receptor inducers; platelet aggregation inhibitors, forexample glycoprotein IIb/IIIa fibrinogen receptor antagonists andaspirin; human peroxisome proliferator activated receptor gamma (PPARγ)agonists, including the compounds commonly referred to as glitazones,for example troglitazone, pioglitazone and rosiglitazone and includingthose compounds included within the structural class known asthiazolidinediones as well as those PPARγ agonists outside thethiazolidinedione structural class; PPARα agonists such as clofibrate,fenofibrate including micronized fenofibrate, and gemfibrozil; PPAR dualα/γ agonists such as5-[(2,4-dioxo-5-thiazolidinyl)methyl]-2-methoxy-N-[[4-(trifluoromethyl)phenyl]methyl]-benzamide,known as KRP-297; vitamin B6 (also known as pyridoxine) and thepharmaceutically acceptable salts thereof such as the HCl salt; vitaminB12 (also known as cyanocobalamin); folic acid or a pharmaceuticallyacceptable salt or ester thereof such as the sodium salt and themethylglucamine salt; anti-oxidant vitamins such as vitamin C and E andbeta carotene; beta-blockers; angiotensin IT antagonists such aslosartan; angiotensin converting enzyme inhibitors such as enalapril andcaptopril; calcium channel blockers such as nifedipine and diltiazam;endothelian antagonists; agents that enhance ABC 1 gene expression; FXRand LXR ligands including both inhibitors and agonists; bisphosphonatecompounds such as alendronate sodium; and cyclooxygenase-2 inhibitorssuch as rofecoxib and celecoxib.

In another embodiment described herein, methods for treatment ofLPA-dependent or LPA-mediated conditions or diseases, such as thetherapy of stroke, comprises administration to a patient compounds,pharmaceutical compositions, or medicaments described herein incombination with at least one additional agent selected from, by way ofexample only, COX-2 inhibitors; nitric oxide synthase inhibitors, suchas N-(3-(aminomethyl)benzyl) acetamidine; Rho kinase inhibitors, such asfasudil; angiotension II type-1 receptor antagonists, includingcandesartan, losartan, irbesartan, eprosartan, telmisartan andvalsartan; glycogen synthase kinase 3 inhibitors; sodium or calciumchannel blockers, including crobenetine; p38 MAP kinase inhibitors,including SKB 239063; thromboxane AX-synthetase inhibitors, includingisbogrel, ozagrel, ridogrel and dazoxiben; statins (HMG CoA reductaseinhibitors), including lovastatin, simvastatin, dihydroxy open-acidsimvastatin, pravastatin, fluvastatin, atorvastatin, nisvastatin, androsuvastatin; neuroprotectants, including free radical scavengers,calcium channel blockers, excitatory amino acid antagonists, growthfactors, antioxidants, such as edaravone, vitamin C, TROLOX™, citicolineand minicycline, and reactive astrocyte inhibitors, such as(2R)-2-propyloctanoic acid; beta andrenergic blockers, such aspropranolol, nadolol, timolol, pindolol, labetalol, metoprolol,atenolol, esmolol and acebutolol; NMDA receptor antagonists, includingmemantine; NR2B antagonists, such as traxoprodil; 5-HTIA agonists;receptor platelet fibrinogen receptor antagonists, including tirofibanand lamifiban; thrombin inhibitors; antithrombotics, such as argatroban;antihypertensive agents, such as enalapril; vasodilators, such ascyclandelate; nociceptin antagonists; DPIV antagonists; GABA 5 inverseagonists; and selective androgen receptor modulators.

In another embodiment described herein, methods for treatment ofLPA-dependent or LPA-mediated conditions or diseases, such as thetherapy of interstitial cystitis, comprises administration to a patientcompounds, pharmaceutical compositions, or medicaments described hereinin combination with at least one additional agent selected from, by wayof example only, dimethylsulfoxide, omalizumab, and pentosanpolysulfate.

In yet another embodiment described herein, methods for treatingLPA-dependent or LPA-mediated conditions or diseases, such as thetherapy of respiratory disorders (e.g., asthma, COPD and rhinitis),comprises administration to a patient compounds, pharmaceuticalcompositions, or medicaments described herein in combination with atleast one agent used in the treatment of respiratory conditions. Agentsused in the treatment of respiratory conditions include, but are notlimited to, bronchodilators (e.g., sympathomimetic agents and xanthinederivatives), leukotriene receptor antagonists, leukotriene formationinhibitors, leukotriene modulators, nasal decongestants, respiratoryenzymes, lung surfactants, antihistamines (e.g., mepyramine(pyrilamine), antazoline, diphenhydramine, carbinoxamine, doxylamine,clemastine, dimenhydrinate, pheniramine, chlorphenamine(chlorpheniramine), dexchlorpheniramine, brompheniramine, triprolidine,cetirizine, cyclizine, chlorcyclizine, hydroxyzine, meclizine,loratadine, desloratidine, promethazine, alimemazine (trimeprazine),cyproheptadine, azatadine, ketotifen, acrivastine, astemizole,cetirizine, mizolastine, terfenadine, azelastine, levocabastine,olopatadine, levocetirizine, fexofenadine), mucolytics, corticosteroids,anticholinergics, antitussives, analgesics, expectorants, albuterol,ephedrine, epinephrine, fomoterol, metaproterenol, terbutaline,budesonide, ciclesonide, dexamethasone, flunisolide, fluticasonepropionate, triamcinolone acetonide, ipratropium bromide,pseudoephedrine, theophylline, montelukast, zafirlukast, ambrisentan,bosentan, enrasentan, sitaxsentan, tezosentan, iloprost, treprostinil,pirfenidone, 5-lipoxygenase-activating protein (FLAP) inhibitors, FLAPmodulators and 5-LO inhibitors.

In a specific embodiment described herein, methods for treatingLPA-dependent or LPA-mediated conditions or diseases, such as thetherapy of asthma and/or COPD, comprises administration to a patientanti-inflammatory agents. In certain embodiments, methods for treatingLPA-dependent or LPA-mediated conditions or diseases, such as thetherapy of asthma and/or COPD, comprise administration to a patientcompounds, pharmaceutical compositions, or medicaments described hereinin combination with at least one additional agent selected from, but notlimited to, epinephrine, isoproterenol, orciprenaline, bronchodilators,glucocorticoids, leukotriene modifiers, mast-cell stabilizers,xanthines, anticholinergics, β-2 agonists, FLAP inhibitors, FLAPmodulators or 5-LO inhibitors. β-2 agonists include, but are not limitedto, short-acting β-2 agonists (e.g., salbutamol (albuterol),levalbuterol, terbutaline, pirbuterol, procaterol, metaproterenol,fenoterol and bitolterol mesylate) and long-acting β-2 agonists (e.g.,salmeterol, formoterol, bambuterol and clenbuterol). FLAP inhibitorsand/or FLAP modulators include, but are not limited to,3-[3-tert-butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionicacid,3-[3-tert-butylsulfanyl-1-[4-(6-ethoxy-pyridin-3-yl)-benzyl]-5-(5-methyl-pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionicacid, MK-886, MK-0591, BAY-x1005 and compounds found in US 2007/0225285,US 2007/0219206, US 2007/0173508, US 2007/0123522 and US 2007/0105866(each of which are hereby incorporated by reference). Glucocorticoidsinclude, but are not limited to, beclometasone, budesonide, ciclesonide,fluticasone and mometasone. Anticholinergics include, but are notlimited to, ipratropium and tiotropium. Mast cell stabilizers include,but are not limited to, cromoglicate and nedocromil. Xanthines include,but are not limited to, amminophylline, theobromine and theophylline.Leukotriene antagonists include, but are not limited to, montelukast,tomelukast, pranlukast and zafirlukast. 5-LO inhibitors include, but arenot limited to, zileuton, VIA-2291 (ABT761), AZ-4407 and ZD-2138 andcompounds found in US 2007/0149579, WO2007/016784.

In another specific embodiment described herein, methods for treatingLPA-dependent or LPA-mediated conditions or diseases, such as thetherapy of allergic diseases or conditions, comprises administration toa patient compounds, pharmaceutical compositions, or medicamentsdescribed herein in combination with at least one additional agentselected from, by way of example only, antihistamines, leukotrieneantagonists, corticosteroids and decongestants. Leukotriene antagonistsinclude, but are not limited to, montelukast, tomelukast, pranlukast andzafirlukast.

In one aspect, LPA receptor antagonists described herein areadministered in combination with one or more agents used to treat usedto treat asthma, including, but not limited to: combination inhalers(fluticasone and salmeterol oral inhalation (e.g. Advair)); inhaledBeta-2 agonists (albuterol inhaler; albuterol nebulizer solution;formoterol; isoproterenol oral inhalation; levalbuterol; metaproterenolinhalation; pirbuterol acetate oral inhalation; salmeterol aerosolinhalation; salmeterol powder inhalation; terbutaline inhaler); inhaledcorticosteroids (beclomethasone oral inhalation; budesonide inhalationsolution; budesonide inhaler; flunisolide oral inhalation; fluticasoneinhalation aerosol; fluticasone powder for oral inhalation; mometasoneinhalation powder; triamcinolone oral inhalation); leukotriene modifiers(montelukast; zafirlukast; zileuton); mast cell stabilizers (cromolyninhaler, nedocromil oral inhalation); monoclonal antibodies(omalizumab); oral Beta-2 agonists (albuterol oral syrup; albuterol oraltablets; metaproterenol; terbutaline); bronchodilator (aminophylline;oxtriphylline; theophylline).

In one aspect, LPA receptor anatogonists described herein areadministered in combination with one or more agents used to treatallergy, including, but not limited to: antihistamine and decongestantcombinations (cetirizine and pseudoephedrine; desloratadine andpseudoephedrine ER; fexofenadine and pseudoephedrine; loratadine andpseudoephedrine); antihistamines (azelastine nasal spray;brompheniramine; brompheniramine oral suspension; carbinoxamine;cetirizine; chlorpheniramine; clemastine; desloratadine;dexchlorpheniramine ER; dexchlorpheniramine oral syrup; diphenhydramineoral; fexofenadine; loratadine; promethazine); decongestants(pseudoephedrine); leukotriene modifiers (montelukast; montelukastgranules); nasal anticholinergics (ipratropium); nasal corticosteroids(beclomethasone nasal inhalation; budesonide nasal inhaler; flunisolidenasal inhalation; fluticasone nasal inhalation; mometasone nasal spray;triamcinolone nasal inhalation; triamcinolone nasal spray); nasaldecongestants (phenylephrine); nasal mast cell stabilizers (cromolynnasal spray).

In one aspect, LPA receptor anatogonists described herein areadministered in combination with one or more agents used to treatchronic obstructive pulmonary disease (COPD), including, but not limitedto: anticholinergics—ipratropium bromide oral inhalation); combinationInhalers (albuterol and ipratropium (e.g. Combivent, DuoNeb);fluticasone and salmeterol oral inhalation (e.g. Advair));corticosteroids (dexamethasone tablets; fludrocortisone acetate;hydrocortisone tablets; methylprednisolone; prednisolone liquid;prednisone oral; triamcinolone oral); inhaled Beta-2 Agonists (albuterolinhaler; albuterol nebulizer solution; formoterol; isoproterenol oralinhalation; levalbuterol; metaproterenol inhalation; pirbuterol acetateoral inhalation; salmeterol aerosol inhalation; salmeterol powderinhalation; terbutaline inhaler); inhaled Corticosteroids(beclomethasone oral inhalation; budesonide inhalation solution;budesonide inhaler; flunisolide oral inhalation; fluticasone inhalationaerosol; fluticasone powder for oral inhalation; triamcinolone oralinhalation); mukolytics (guaifenesin); oral Beta-2 agonists (albuteroloral syrup; albuterol oral tablets; metaproterenol; terbutaline);bronchodilator (aminophylline; oxtriphylline; theophylline).

In one embodiment, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is administered to a patient in combinationwith inhaled corticosteroids.

In one embodiment, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is administered to a patient in combinationwith beta2-adrenergic receptor agonists. In one embodiment, Compound 1,or a pharmaceutically acceptable salt thereof (e.g. Compound 2) isadministered to a patient in combination with short actingbeta2-adrenergic receptor agonists. In one embodiment. Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2) isadministered to a patient in combination with long-actingbeta2-adrenergic receptor agonists.

In one embodiment, Compound 1, or a pharmaceutically acceptable saltthereof (e.g. Compound 2) is combined with or administered incombination with one or more agents that are inhibitors ofUDP-glucuronosyltransferase (UGT). UGT inhibitors include thosedescribed in U.S. 2003/0215462; U.S. 2004/0014648. In some embodiments,co-administration of a UGT inhibitor allows for lower doses of Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2) to beadministered.

The individual compounds of such combinations are administered eithersequentially or simultaneously in separate or combined pharmaceuticalformulations. In one embodiment, the individual compounds will beadministered simultaneously in a combined pharmaceutical formulation.Appropriate doses of known therapeutic agents will be appreciated bythose skilled in the art.

The combinations referred to herein are conveniently presented for usein the form of a pharmaceutical compositions together with apharmaceutically acceptable diluent(s) or carrier(s).

Kits/Articles of Manufacture

For use in the therapeutic methods of use described herein, kits andarticles of manufacture are also described herein. Such kits include acarrier, package, or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in amethod described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. In one embodiment, thecontainers are formed from a variety of materials such as glass orplastic.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical productsinclude, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment. A wide array offormulations of the compounds and compositions provided herein arecontemplated as are a variety of treatments for any disease, disorder,or condition that would benefit by antagonism of LPA receptors.

For example, the container(s) include Compound 1, or a pharmaceuticallyacceptable salt thereof (e.g. Compound 2), optionally in a compositionor in combination with another agent as disclosed herein. Such kitsoptionally include an identifying description or label or instructionsrelating to its use in the methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

It is to be understood that as used herein, pharmaceutical compositionsdescribed as comprising a pharmaceutically acceptable salt describedherein, e.g., liquid solutions, encompass pharmaceutical compositionscomprising the associated and/or disassociated forms of the salt. Thus,for example, a pharmaceutical composition described herein comprising anaqueous solution of Compound 2 encompasses a composition comprising apopulation of sodium cations and a population of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylateanions.

EXAMPLES

The following ingredients, formulations, processes and procedures forpracticing the methods disclosed herein correspond to that describedabove. The procedures below describe with particularity illustrative,non-limiting embodiment of formulations that include a Compound 1, or apharmaceutically acceptable salt and/or solvate thereof, andpharmacokinetic profiles and pharmacodynamic effects thereof. By way ofexample only, Compound 1 is optionally prepared as outlined in U.S.patent application Ser. No. 12/793,440, or as outlined herein.

Example 1: Synthesis of1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1) Step 1: 3-Methylamino-but-2-enoic acid methyl ester

To a solution of methyl acetoacetate (29.4 g, 253 mmol) in MeOH (30 mL)was added methylamine (33 wt % in EtOH; 48 mL, 385 mmol) dropwise atroom temperature. The reaction was stirred for 1 hour, and thenconcentrated and dried to give the title compound as a white crystallinesolid.

Step 2: 2-(4-Bromo-benzoyl)-3-oxo-butyric acid methyl ester

To 3-methylamino-but-2-enoic acid methyl ester (5.0 g, 39.1 mmol) in THF(70 mL) was added pyridine (3.7 mL). The mixture was cooled to 0° C.,and 4-bromobenzoyl chloride (8.55 g, 39.1 mmol) in THF (30 mL) was addeddropwise over 2 minutes. The reaction was warmed to room temperatureover 1 hour and then stirred at room temperature overnight. Aqueouswork-up gave the title compound.

Step 3: 5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid methylester

2-(4-Bromo-benzoyl)-3-oxo-butyric acid methyl ester (11 g, 39 mmol) andhydroxylamine hydrochloride (2.66 g, 39 mmol) were combined in aceticacid (50 mL), and the reaction was stirred at 115° C. for 1 hour. Aftercooling, aqueous work-up gave the title compound.

Step 4: 5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid

Lithium hydroxide (2 g, 47.7 mmol) was added to a solution of5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid methyl ester (7g, 23.6 mmol) in MeOH (50 mL) and H₂O (10 mL), and the reaction wasstirred at 60° C. for 1 hour. Acidic work-up the title compound.

Step 5: [5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid(R)-1-phenyl-ethyl ester

5-(4-Bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid (2.0 g, 7.09mmol) and triethylamine (0.99 mL, 7.09 mmol) were dissolved in toluene(50 mL). Diphenylphosphoryl azide (1.5 mL, 7.09 mmol) was added,followed by (R)-(+)-1-phenylethyl alcohol (0.865 g, 7.09 mmol;commercially available or prepared using procedures described herein orin the literature: e.g. E. J. Corey et al. J. Am. Chem. 1987, 109,5551-5553), and the reaction was stirred at 80° C. for 4 hours. Themixture was concentrated, and the residue was purified by silica gelchromatography to give the title compound.

Step 6:1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid

[5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid(R)-1-phenyl-ethyl ester (0.248 g, 0.62 mmol),4-(1′-carboxyl-cyclopropyl)phenylboronic acid (0.160 g, 0.62 mmol), andsodium carbonate (0.155 g, 1.85 mmol) were combined in 2:1 DME:H₂O. Thesolution was purged with N₂ for 10 minutes, and thenbis(triphenylphosphine)palladium(II) dichloride (0.047 g, 0.06 mmol) wasadded. The reaction was purged with N₂ for an additional 10 minutes, andthen stirred in a sealed tube at 80° C. for 2 hours. The mixture waspartitioned between EtOAc and H₂O, and the aqueous layer was extractedwith EtOAc. The combined organic layers were dried over MgSO₄, filtered,and concentrated, and the residue was purified by silica gelchromatography to give the title compound. Mass spec. data (M+H)=483.

Example 2: Alternate synthesis of1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1) Step 1: 1-(biphenyl-4-yl)cyclopropanecarbonitrile

4-phenyl-phenylacetonitrile (VWR scientific, 100 g, 518 mmol) was addedto a solution of KOH (174 g, 3.1 mol) in water (175 mL) and toluene (500mL) at room temperature. Tetrabutyl ammonium bromide (8.33 g, 26 mmol)followed by 1,2 dibromoethane (116.3 g, 622 mmol) were added and thesolution was heated to 60° C. for 4 hours. Add 10 mL dibromoethane andcontinue heating at 60° C. for another 20 hours. Reaction isapproximately 50% complete. Add KOH (116 g, 622 mmol), dibromoethane (20mL) and tetrabutyl ammonium bromide (8.33 g, 26 mmol) and hcat to 80° C.for an additional 24 hours. Reaction complete by TLC (20% EtOAc/hex).The organic layer was extracted with water (500 mL) one time and dilutehydrochloric acid (500 mL, pH ˜3) one time. The organic layer wasevaporated to yield product.

Step 2: 1-(Biphenyl-4-yl)cyclopropanecarboxylic acid:1-(Biphenyl-4-yl)cyclopropanecarbonitrile

(112 g, 511 mmol), KOH (114 g, 2.04 mol) and ethylene glycol (400 mL)were heated to 170° C. for 3 hours. The solution was cooled to roomtemperature, poured into water (900 mL) and the solution acidified with˜150 mL conc. HCl (slowly) to precipitate the product. The product wasfiltered and washed with 500 mL water. The solid was resuspended inwater (800 mL), stirred for ˜15 minutes and filtered. The resulting wetsolid was dried in a vacuum over overnight at 80° C. to yield product.

Step 3: 1-(Biphenyl-4-yl)cyclopropanecarboxylic acid ethyl ester

1-(Biphenyl-4-yl)cyclopropanecarboxylic acid (116 g, 487 mmol), ethanol(400 mL) and sulfuric acid (50 mL) were heated to reflux for 16 hours.The product was extracted with CH₂Cl₂ (500 mL) and water (800 mL) dried,filtered and evaporated to yield product.

Step 4: 1-(4′-Acetylblphenyl-4-yl)cyclopropanecarboxylic acid ethylester

To 1-(biphenyl-4-yl)cyclopropanecarboxylic acid ethyl ester (90 g, 376mmol) in CH₂Cl₂ (450 mL) was added acetyl chloride (31.7 g, 406 mmol)followed by aluminum chloride (94.5 g, 710 mmol) over ˜30 minutes. Thesolution was stirred at room temperature for 2 hours. The reaction wasslowly poured into 1 M HCl (500 mL) and the organic layer separated. Theorganic layer was washed 2 times with water (500 mL), dried (MgSO4),filtered and evaporated to yield product.

Step 5: 4′-(1-(Ethoxycarbonyl)cyclopropyl)biphenyl-4-carboxylic acid

To 1-(4′-acetylbiphenyl-4-yl)cyclopropanecarboxylic acid ethyl ester(10.1 g, 33 mmol) in dioxane (200 mL) at ˜10° C. was added a solution ofbromine (26.4 g, 165 mmol), sodium hydroxide (22.4 g, 561 mmol) in water(150 mL). The solution was stirred at room temperature for 30 minutes,poured into water (500 mL) and acidified with dilute hydrochloric acid.Sodium metabisulfite was added until the brown bromine color dissipated.The product was filtered and dried in a vacuum over overnight at 40° C.to yield 10 g of 4′-(1-(ethoxycarbonyl)cyclopropyl)biphenyl-4-carboxylicacid.

Step 6: 3-Methylamino-but-2-enoic acid benzyl ester

To benzyl acetoacetate (29 g, 151 mmol) in ethanol (30 mL) was addedmethyl amine (33% in ethanol, 7.02 g, 226 mmol). The solution wasstirred for 2 hours at room temperature followed by evaporation to yielda yellow oil.

Step 7: Ethyl1-(4′-(2-(benzyloxycarbonyl)-3-(methylamino)but-2-enoyl)biphenyl-4-yl)cyclopropanecarboxylate

4′-(1-(Ethoxycarbonyl)cyclopropyl)biphenyl-4-carboxylic acid (80 g, 258mmol), dichloroethane (400 mL), DMF (0.1 mL), thionyl choride (2.3 mL,32 mmol) were heated to 80° C. for 1.5 hours. (acid chloride formationwas monitored by adding small aliquot (100 μL) to a solution of benylamine in acetonitrile and analyzing for the benzyl amide by LCMS; nostarting material was observed by LCMS). The solution was evaporated ona rotavap to a dark oil and a solution of eneamine (68.4 g, 335 mmol),pyridine (44.8 g, 568 mmol) in THF (400 mL) was added. The solution wasstirred at 50° C. for 2 hours then the volatiles were evaporated using arotavap to yield the crude product as a dark semi-solid.

Step 8: Benzyl5-(4′-(1-(ethoxycarbonyl)cyclopropyl)biphenyl-4-yl)-3-methylisoxazole-4-carboxylate

To the crude material from the previous reaction was added hydroxylamine hydrochloride (26.7 g, 387 mmol) and acetic acid (400 mL). Thesolution was heated to 95° C. for 1 hour cooled to room temperature,extracted with CH₂Cl₂ and water 3 times, dried, evaporated. The crudeproduct is purified by running through a plug of silica (˜200 grams ofSiO2) eluting with CH2Cl2, then recrystallized in ethanol to yieldproduct.

Step 9:5-(4′-(1-(ethoxycarbonyl)cyclopropyl)biphenyl-4-yl)-3-methylisoxazole-4-carboxylicacid

The benzyl ester (54 g, 112 mmol) in THF (300 mL) was degassed withnitrogen for 20 minutes, 10% Palladium on activated carbon (1.2 g, 1.1mmol) was added and the solution was sparged with hydrogen via balloon.The balloon of hydrogen was maintained on the head space and thesolution stirred for 20 hours. The reaction was filtered through celiteand evaporated to dryness. The solid was triturated with a 1/1 solutionof hexane/ethyl acetate (˜300 mL) and filtered to yield product.Evaporation of the mother liquor followed by trituration of the solidwith 1/1 hexanes ethyl acetate yielded further product.

Step 10:1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid ethyl ester

To the acid from Step 9 (0.5 g, 1.28 mmol) in toluene (5 mL) was added(R)-1-phenyl ethanol (0.16 g, 1.34 mmol), triethyl amine (0.26 g, 2.56mmol) and diphenyl phosphoryl azide (0.39 g, 1.4 mmol). The solution washeated to 80° C. for 1 hour, cooled to room temperature and extractedwith water 3 times. The organic layer wad dried and evaporated to yield0.61 g. The product was further purified by column 0 to 40% EtOAc/hex toyield product.

Step 11:1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid

To ethyl ester (22.7 g, 44 mmol) in methanol (300 mL) was added lithiumhydroxide (9.1 g, 222 mmol). The solution was heated to 65° C. for 2hours, extracted into methylene choride and washed with dilutedhydrochloric acid. The organic layer was dried and evaporated to yieldproduct.

Example 3: Alternate synthesis of1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1) Step 1: 1-(Biphenyl-4-yl)cyclopropanecarboxylic acidisopropyl ester

1-(Biphenyl-4-yl)cyclopropanecarboxylic acid (10 g, 42 mmol),isopropanol (100 mL), thionyl chloride (6.8 mL, 92 mmol) were heated to65° C. for 4 hours. Sulfuric acid (20 mL) was added and heated at 65° C.overnight. The product is extracted with CH₂Cl₂ and water (2×) dried andevaporated to yield product.

Step 2: 1-(4′-Acetylbiphenyl-4-yl)cyclopropanecarboxylic acid isopropylester

To 1-(biphenyl-4-yl)cyclopropanecarboxylic acid isopropyl ester (10.2 g,36 mmol) in CH₂Cl₂ (100 mL) was added aluminum chloride (10.2 g, 76.5mmol) followed by acetyl chloride (5.97 g, 76.5 mmol). The solution wasstirred at room temperature for 1.5 hours then slowly poured into water.The organic layer was separated and extracted 1 time with sodiumpotassium tartrate solution (20 g in 250 mL water). The organic layerwas dried and evaporated to yield product.

Step 3: 4′-(1-(isopropoxycarbonyl)cyclopropyl)biphenyl-4-carboxylic acid

To 1-(4′-acetylbiphenyl-4-yl)cyclopropanecarboxylic acid isopropyl ester(11.6 g, 36 mmol) in dioxane (200 mL) at ˜10° C. was added a solution ofbromine (28.8 g, 180 mmol), sodium hydroxide (24.5 g, 612 mmol) in water(150 mL). The solution was stirred at room temperature for 30 minutespoured into water (500 mL) and acidified with dilute hydrochloric acid.Sodium metabisulfite was added until the brown bromine color dissipated.The product was filtered and dried in a vacuum over overnight at 40° C.to yield product.

Step 4: Isopropyl1-(4′-(2-(benzyloxycarbonyl)-3-(methylamino)but-2-enoyl)biphenyl-4-yl)cyclopropanecarboxylate

4′-(1-(Isopropoxycarbonyl)cyclopropyl)biphenyl-4-carboxylic acid (9.2 g,28 mmol), dichloroethane (50 mL), DMF (0.1 mL), thionyl choride (5.5 mL,62 mmol) were heated to 75° C. for 1.5 hours. (acid chloride formationwas monitored by adding small aliquot (100 μL) to a solution of benylamine in acetonitrile and analyzing for the benzyl amide by LCMS; nostarting material was observed by LCMS). The solution was evaporated ona rotavap and THF (10 mL) was added. The solution of the acid chloridein THF was added via syringe to a solution of 3-methylamino-but-2-enoicacid methyl ester (4.0 g, 31.2 mmol) and pyridine (5.5 mL, 70 mmol) inTHF (50 mL). The solution was stirred at room temperature overnight. Thevolatiles were evaporated on a rotavap to yield the crude product.

Step 5: Methyl5-(4′-(1-(isopropoxycarbonyl)cyclopropyl)biphenyl-4-yl)-3-methylisoxazole-4-carboxylate

To the crude material from the previous reaction was added hydroxylamine hydrochloride (2.9 g, 42 mmol) and acetic acid (50 mL). Thesolution was heated to 100° C. for 30 minutes cooled to roomtemperature, extracted with CH₂Cl₂ and water (4 times, second and thirdtime made basic with sodium bicarbonate). The organic phase was dried,evaporated and purified on column (220 g silica; 0 to 20% EtOAc/hexanes)to yield product.

Step 6:5-(4′-(1-(propoxycarbonyl)cyclopropyl)biphenyl-4-yl)-3-methylisoxazole-4-carboxylicacid

To the methyl ester from Step 5 (5.2 g, 12.4 mmol) in THF (100 mL) andethanol (20 mL) was added a solution of sodium hydroxide (1.5 g, 37.2mmol) in water (40 mL). The solution was stirred at room temperature 3hours. ˜50 mL solvent evaporated and 200 mL water added. The product wasprecipitated out of solution with dilute hydrochloric acid to pH 2. Theproduct was isolated by filtration to yield product.

Step 7:1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid isopropyl ester

To the acid from Step 6 (4.0 g, 10 mmol) in toluene (50 mL) was addedR-1-pheynyl ethanol (1.33 g, 11 mmol), triethyl amine (2.02 g, 20 mmol)and diphenyl phosphoryl azide (3.16 g, 11.5 mmol). The solution washeated to 80° C. for 1 hour cooled to room temperature and extractedwith water 3 times. The organic layer wad dried and evaporated to yieldproduct.

Step 8:1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid

To the isopropyl ester from Step 7 (5.2 g, 10 mmol) in THF (30 mL), MeOH(10 mL) was added NaOH (2 g, 50 mmol) in water (10 mL). The solution isheated to 65° C. for 5 hours. The solution was cooled to roomtemperature, extracted with methylene chloride and dilute hydrochloricacid. The organic was dried and evaporated and the product was purifiedby column chromatography (0 to 60% EtOAc/hexanes) to yield product.

Example 4: Synthesis of1-[4-(0,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-cyclopropanecarboxylicacid ethyl ester Step 1: 1-(4-Bromo-phenyl)-cyclopropanecarbonitrile

Potassium hydroxide (14.3 g, 255 mmol) was dissolved in H₂O (5 mL) andtoluene (40 mL), 4-Bromophenylacetonitrile (5.0 g, 25.5 mmol) andtetrabutylammonium bromide (0.41 g, 1.3 mmol) was added, followed by1,2-dibromoethane (3.25 mL, 38 mmol) dropwise over 10 minutes. Thereaction was stirred at room temperature for 2 hours and then worked-upto give the title compound.

Step 2: 1-(4-Bromo-phenyl)-cyclopropanecarboxylic acid

1-(4-Bromo-phenyl)-cyclopropanecarbonitrile (5 g, 22.5 mmol) andpotassium hydroxide (5 g, 89.3 mmol) were combined in ethylene glycol(70 mL), and the reaction was stirred at 180° C. for 4 hours. Themixture was poured into H₂O, acidified, and filtered to give the titlecompound.

Step 3: 1-(4-Bromo-phenyl)-cyclopropanecarboxylic acid ethyl ester

1-(4-Bromo-phenyl)-cyclopropanecarboxylic acid (5 g, 20.7 mmol) in EtOH(50 mL) was treated with sulfuric acid (2 mL), and the reaction wasstirred at 75° C. for 1 hour. The mixture was worked up to give thetitle compound.

Step 4:1-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-cyclopropanecarboxylicacid ethyl ester

1-(4-Bromo-phenyl)-cyclopropanecarboxylic acid ethyl ester (3.6 g, 13.4mmol), bis(pinacolato)diboron (3.37 g, 16.1 mmol), and potassium acetate(2.8 g, 29 mmol) were combined in 1,4-dioxane (30 mL). The solution waspurged with N₂ for 10 minutes, and then(1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium(II) (0.50 g,0.65 mmol) was added and the reaction was heated to 80° C. for 2 hours.Aqueous work-up, followed by silica gel chromatography (0-30% EtOAc inhexanes), gave the title compound.

Example 5: Synthesis of1-{4′-[3-Methyl-4-((S)-1-phenyl-ethoxycarbonylamino-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid Step 1: (S)-[5-(4-Bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamicacid 1-phenyl-ethyl ester

Prepared according to the procedure described in Example 1, Step 5 usingthe following starting materials:5-(4-bromo-phenyl)-3-methyl-isoxazole-4-carboxylic acid and(S)-(−)-1-phenylethanol (commercially available or prepared usingprocedures described herein or in the literature: e.g. E. J. Corey etal. J. Am. Chem. 1987, 109, 5551-5553).

Step 2:1-{4′-[3-Methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid ethyl ester

Prepared according to the procedure described in Example 1, Step 6 usingthe following starting materials:(S)-[5-(4-bromo-phenyl)-3-methyl-isoxazol-4-yl]-carbamic acid1-phenyl-ethyl ester and1-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-cyclopropanecarboxylicacid ethyl ester.

Step 3:1-{4′-[3-Methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-Isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid

To1-{4′-[3-methyl-4-((S)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid ethyl ester (1 equivalent) in 2:1 MeOH:H₂O was added lithiumhydroxide (3-10 equivalents), and the reaction was stirred at roomtemperature until no starting material was seen by analytical LCMS. Themixture was acidified with IN aqueous HCl and extracted with EtOAc. Thecombined organic layers were dried, filtered, and concentrated to givethe title compound. Mass spec. data (M+H)=483.

Example 6: Synthesis of racemic1-{4′-[3-Methyl-4-(1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid

Prepared in a similar manner to the procedures described in Example 1using (R/S)-1-phenyl-ethanol in place of (R)-1-phenyl-ethanol. Massspec. data (M+H)=483.

Example 7: Synthesis of1-{4′-[3-Methyl-4-(1-phenyl-ethoxy-d9-carbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid Step 1:1-{4′-[3-Methyl-4-(1-phenyl-ethoxy-d9-carbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid ethyl ester

Prepared according to the procedure described in Example 1, Step 5 usingthe following starting materials:5-[4′-(1-ethoxycarbonyl-cyclopropyl)-biphenyl-4-yl]-3-methyl-isoxazole-4-carboxylicacid and 1-phenylethanol-d9 (obtained from Carbocore).

Step 2:1-{4′-[3-Methyl-4-(1-phenyl-ethoxy-d9-carbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid

To1-{4′-[3-methyl-4-(1-phenyl-ethoxy-d9-carbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid ethyl ester (1 equivalent) in 2:1 MeOH:H₂O was added lithiumhydroxide (3-10 equivalents), and the reaction was stirred at roomtemperature until no starting material was seen by analytical LCMS. Themixture was acidified with IN aqueous HCl and extracted with EtOAc. Thecombined organic layers were dried, filtered, and concentrated to givethe title compound. Mass spec. data (M+H)=492.

In some embodiments, Mass spectrometric data (mass spec. data) isobtained on with a Shimadzu LCMS 2010A.

Example 8: Preparation of Crystalline1-{4′-[3-Methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1)

20 mg of Compound 1 was weighed into a HPLC vial. 40 μL of ethanol wasadded and the vial was heated to reflux with a hot air gun. The solutionwas cooled to ambient temperature and the solids were filtered off.Crystalline Compound 1 (Pattern 1) was obtained as determined by XRPD.In the same manner as described from ethanol, the following solventsalso provided crystalline Compound 1 (Pattern 1): methanol,2-methoxyethanol, ethanol, 1-propanol, 2-propanol, 1-butanol, butylacetate, acetone, methylethyl ketone, anisole, toluene. 5 Volumes (i.e.100 μL) of the following solvents with heating also provided crystallineCompound 1 (Pattern 1): nitromethane, acetonitrile, ethyl acetate,cumene.

Compound 1 dissolved in 2 volumes (i.e. 40 μL) of the following solventsat ambient temperature: 1-4-dioxane, tetrahydrofuran. The vials wereuncapped and allowed to slowly evaporate. This provided crystallineCompound 1 (Pattern 1).

In another embodiment, 4.6 mg of Compound 1 was dissolved in 0.2 mlacetonitrile and 0.1 ml H₂O. The resulting solution was covered andslowly evaporated at 50° C. Need-shaped crystals were obtained from thesolution after 5 days. This provided crystalline Compound 1 (Pattern 1).

In another embodiment, 4.9 mg of Compound 1 was dissolved in 0.2 mlisopropyl alcohol and 0.1 ml H₂O. The resulting solution was covered andslowly evaporated at 50° C. Need-shaped crystals were obtained from thesolution after 3 days. This provided crystalline Compound 1 (Pattern 1).

In one embodiment, 4.5 mg of Compound 1 was dissolved in 0.2 ml methanoland 0.05 ml H₂O. The resulting solution was covered and slowlyevaporated at 50° C. Need-shaped crystals were obtained from thesolution after 3 days. This provided crystalline Compound 1 (Pattern 2).In another embodiment, 115 mg of Compound 1 was dissolved in 0.98 mL ofmethanol and 0.12 mL of tetrahydrofuran. The solution was evaporated todryness and then the following was charged: a) 1 mL of acetonitrile and1 mL of water; or b) 0.5 mL of ethanol, 0.5 mL of propylene glycol and 1mL of heptanes; or c) 1 mL of ethyl acetate and 1 mL of heptanes; or d)1 mL of methyl isobutyl ketone and 1 mL of heptanes. The crystallizationwas then stirred for 72 hours. This provided Compound 1 (Pattern 2).

In another embodiment, 305 mg of Compound 2 was dissolved in 10.0 mlTHF. This solution was dispensed to a high throughput crystallizationplate and test solvents were added. Needle-shaped crystals wererecovered from the well which consisted of the solvent mixture of 1:2THF and H₂O. This provided crystalline Compound 1 (Pattern 3).

In another embodiment, 2.2 mg of Compound 1 was dissolved in 0.15 mlmethanol and 0.05 ml H₂O. The resulting solution was covered and storedat ambient conditions. Need-shaped crystals were obtained from thesolution after 2 days. This provided crystalline Compound 1 (Pattern 3).

Compound 1 exhibits good solubility in a range of solvents, with onlywater and highly non-polar solvents (e.g. cyclohexane, heptane) provingunsuitable.

Example 9: Preparation of Compound 2 from Compound 1

Compound 1 was suspended in 10 volumes of ethanol and 1.0 equivalent 50wt. % sodium hydroxide was added. Then heptane (20 vol.) was added overa 2-4 hour period. The ethanol was removed in vacuum and solventexchanged with heptane. The solid was collected under nitrogen by vacuumfiltration and the product was dried at about 60° C.-100° C. in a vacuumoven. This procedure gave Compound 2 in high purity.

Example 10: Preparation of Crystalline Compound 2

Maturation of Compound 2

100 mg of Compound 2 was weighed into five vials. To each vial was addeda separate solvent: 1000 μL of nitormethane; 1000 μL of acetonitrile;500 μL of 2-propanol; 500 μL of 1-butanol; 1000 μL of anisole. The vialswere then cycled between ambient and 50° C., with 4 hour periods at eachtemperature, for 4 days. Any solids present at this time were filteredoff and analysed by XRPD. Solids were then dried under vacuum at 50° C.for 2 days and reanalysed by XRPD. Amorphous material was obtained fromnitromethane. Partially crystalline material was obtained from2-propanol, 1-butanol and anisole. Crystalline compound 2 (Pattern 1)was isolated from acetonitrile.

The solids obtained from 1-butanol were noted to contain ⅔ of anequivalent of 1-butanol by ¹H NMR. TGA analysis was also performed and amass loss observed up to 160° C., when taken with the 1H NMR data,indicate that a 1-butanol solvate of the sodium salt was obtained. A12.43% mass loss in the TGA equates to 0.92 equivalents of 1-butanolbeing present in the sample.

Anti-Solvent Mediated Conditions

20 mg of Compound 2 was weighed into HPLC vials. To each vial 100 uL ofa 1:1 mixture of a solvent:anti-solvent was added. The vials were thenagitated at 30° C. for a period of 12 days. After this period any solidswere filtered off and analysed by XRPD.

Experiments using 1,4-dioxane/tert-butyl methyl ether and methyl ethylketone (MEK)/tert-butyl methyl ether produced crystalline compound 2(Pattern 1) with the same diffraction pattern as that isolated from theacetonitrile maturation experiment. Partially crystalline material wasobtained from ethyl acetate/tert-butyl methyl ether andanisole/tert-butyl methyl ether.

Preparation of Hydrated Crystalline Compound 2 (Pattern 1)

16.5 g of amorphous Compound 2, 83 ml (5 vol) of methyl ethyl ketone(MEK) and 4.1 ml (0.25 vol) of water slurried at 60° C. for 30 mins. 165ml (10 vol) of MEK added and the slurry seeded with ca. 15 mg ofCompound 2 (Pattern 1). Slurry cooled to 50° C. for 6 hours and thenfurther cooled to 15° C. over 6 hrs and stirred at ambient for a further10 hrs. Filtered and dried in-vacuo for 21 hours. Recovery=15.7 g (91%).This procedure provided the hydrated crystalline Compound 2 (Pattern 1)with no significant amorphous content. At least 97% pure.

In an alternative procedure, 4.8 mg of Compound 2 was dissolved in 0.2ml acetonitrile and 0.2 ml tetrahydrofuran. The resulting solution wasslowly evaporated at 50° C. Plate-shaped single crystals were recoveredafter 3 days.

Preparation of Crystalline Compound 2 (Pattern 2)

1 g of Compound 1 was added into methyl ethyl ketone (MEK) (5 mL, 5vol). Sodium hydroxide (83 mg) and water (213 mg, 0.21 vol) were added.The solution was heated to 60° C. and MEK (10 ml, 10 vol) was slowlyadded to the solution at 60° C. The reaction mixture was seeded with 10mg of the hydrate crystalline Compound 2 and agitated at 50° C. for 2hours. The cloudy solution was then cooled down to 20° C. over 2 hours.The solid was collected and dried under vacuum at 40° C. for 24 hours.

Preparation of Crystalline Compound 2 (Pattern 3)

Charged to a 50 L multi-neck round bottom flask 31 L methanol (MeOH),3.1 kg Compound 1, and 514.0 g of 50% sodium hydroxide and agitateduntil a complete solution was obtained. Distilled the reactor contentsuntil ˜10 L remained maintaining a jacket temperature of about 45° C.Charged 25.0 kg ethanol to the reactor and distilled until ˜15 Lremained. Charged 25.0 kg ethanol to the reactor and distilled until ˜15L remained. Charged 12.1 kg ethanol to the reactor and agitated with ajacket temperature of 20° C. for at least 20 minutes. Charged 42.1 kgheptane to the reactor over a period of at least 4 hr The reactor wasagitated for 4 hr before distilling until ˜45 L remained in the reactor.32.0 kg of heptane was charged and distilled two (2) times beforecharging 32.1 kg heptane and filtering the reactor contents. The filtercake was washed with 16.0 kg heptane and the cake blown dry. The filtercake was dried in the oven maintaining a temperature of about 65 C. Thedry Compound 2 was charged to the reactor with a pre-made solution of12.6 kg methyl ethyl ketone (MEK), 45.9 kg methyl t-butyl ether (MTBE),and 0.31 kg water. The reactor was agitated at 45±5 C for 16 hr. Thereactor contents were agitated an additional 21.5 hours. 60 g ofCompound 2 seed crystals slurried in 1552 g MTBE. The reactor wasagitated for 52.3 hours. 150 mL water was added. The reactor wasagitated for 11 hr. Total time from start of crystallization tocrystallization deemed complete was 7 days. The reactor was agitated at20±5 C for 1 hr before the reactor contents were filtered. The filtercake was not washed. The filter cake was dried in the oven maintaining atemperature of about 65 C.

The Compound 2 was screened using a 20 mesh screen and returned to theoven to be dried at a temperature of about 85° C. Compound 2 was held inoven for 4 hours.

Example 11: Additional Salts of Compound 1

The free acid (Compound 1; 20 mg) was placed in a HPLC vial and treatedwith acetonitrile (200 μL), ethanol (200 μL), ethyl acetate (200 μL) ortoluene (400 μL). The vial was capped, warmed and shaken until completedissolution was achieved. One equivalent of base was added. Basesolutions included 10M KOH (water); 5M L-arginine (water); 10M L-Lysine(water); 2M NH—OH (28% aqueous solution); or 1M N-Me-glucamine (water).Vials were capped and allowed to stand at room temperature for six days.Any solids formed were filtered off.

Using this procedure, salts of Compound 1 with L-lysine, ammonium andN-methyl-D-glucamine salts were isolated.

Crystalline L-lysine salt of Compound 1 was obtained in ethanol andethyl acetate.

Crystalline ammonium salt of Compound 1 was obtained in toluene.

Crystalline N-methyl-D-glucamine salt of Compound 1 was obtained inacetonitrile.

Example 12: X-Ray Characterization

The crystalline forms were analyzed using one or more of the testingmethods described below. It is understood that slight variations in thecoordinates and peak data for the X-ray measurements are possible andare considered to be within the scope of the present disclosure. In someembodiments, 2-Theta peak values that are provided for the XRPD arewithin ±0.1° 2-Theta.

X-Ray Powder Diffraction (XRPD)

X-Ray powder diffraction patterns were collected on a Bruker AXS C2GADDS or Bruker AXS D8 Advance diffractometer.

Bruker AXS C2 GADDS

X-Ray Powder Diffraction patterns were collected on a Bruker AXS C2GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZstage, laser video microscope for auto-sample positioning and a HiStar2-dimensional area detector. X-ray optics consisted of a single Göbelmultilayer mirror coupled with a pinhole collimator of 0.3 mm. The beamdivergence, i.e. the effective size of the X-ray beam on the sample, wasapproximately 4 mm. A θ-θ continuous scan mode was employed with asample—detector distance of 20 cm which gives an effective 2θ range of3.20°-29.7°. Typically the sample would be exposed to the X-ray beam for120 seconds. The software used for data collection was GADDS for WNT4.1.16 and the data were analyzed and presented using Diffrac Plus EVA v9.0.0.2 or v 13.0.0.2.

Ambient Conditions

Samples run under ambient conditions were prepared as flat platespecimens using powder as received without grinding. Approximately 1-2mg of the sample was lightly pressed on a glass slide to obtain a flatsurface.

Non-Ambient Conditions

Samples run under non-ambient conditions were mounted on a silicon waferwith heat-conducting compound. The sample was then heated to theappropriate temperature at ca. 10° C.·min⁻¹ and subsequently heldisothermally for ca 2 minutes before data collection was initiated.

Bruker AXS D8 Advance

X-Ray Powder Diffraction patterns were collected on a Bruker D8diffractometer using Cu Kα radiation (40 kV, 40 mA), θ−2θ goniometer,and divergence of V4 and receiving slits, a Ge monochromator and aLynxeye detector. The instrument was performance checked using acertified Corundum standard (NIST 1976). The software used for datacollection was Diffrac Plus XRD Commander v2.5.0 and the data wereanalyzed and presented using Diffrac Plus EVA v 11.0.0.2 or v 13.0.0.2.Samples were run under ambient conditions as flat plate specimens usingpowder. Approximately 5 mg of the sample was gently packed into a cavitycut into polished, zero-background (510) silicon wafer. The sample wasrotated in its own plane during analysis. The details of the datacollection are:

-   -   Angular range: 2 to 42° 2θ    -   Step size: 0.05° 2θ    -   Collection time: 0.5 s·step⁻¹

XRPD on Pattern 1 Free Acid (Compound 1)

The X-Ray powder diffraction pattern for Pattern 1 of the free acid(Compound 1) is displayed in FIG. 1. Characteristic peaks include 4.7°2-Theta, 9.4° 2-Theta, 14.5° 2-Theta, and 21.0° 2-Theta.

No form change was noted by XRPD after either GVS analysis or storage at40° C./75% RH for one week.

XRPD on Hydrated Crystalline Compound 2 (Pattern 1)

The X-Ray powder diffraction pattern for Pattern 1 of the sodium salt(Compound 2) is displayed in FIG. 4. Characteristic peaks include 8.5°2-Theta, 13.2° 2-Theta, 17.2° 2-Theta, 19.3° 2-Theta, 22.4° 2-Theta, and25.6° 2-Theta.

XRPD on Crystalline Compound 2 (Pattern 2)

The X-Ray powder diffraction pattern for Pattern 2 of the sodium salt(Compound 2) is displayed in FIG. 8.

XRPD on Crystalline Compound 2 (Pattern 3)

The X-Ray powder diffraction pattern for Pattern 3 of the sodium salt(Compound 2) is displayed in FIG. 9.

XRPD on Amorphous Compound 2

The X-Ray powder diffraction pattern for amorphous sodium salt (Compound2) is displayed in FIG. 10.

XRPD on Crystalline Compound 1 (Pattern 2)

X-ray powder diffraction (XPRD) data were obtained using a Bruker C2GADDS. The radiation was Cu Kα (40 KV, 40 mA). The sample-detectordistance was 15 cm. Powder samples were placed in sealed glasscapillaries of 1 mm or less in diameter; the capillary was rotatedduring data collection. Data were collected for 3≦2θ≦35° with a sampleexposure time of at least 1000 seconds. The resulting two-dimensionaldiffraction arcs were integrated to create a traditional 1-dimensionalXPRD pattern with a step size of 0.02 degrees 2θ in the range of 3 to 35degrees 2θ.

The X-Ray powder diffraction pattern for Pattern 2 of the free acid(Compound 1) is displayed in FIG. 12. Characteristic peaks include 6.3°2-Theta, 12.8° 2-Theta, 16.4° 2-Theta, 17.0° 2-Theta, 19.7° 2-Theta.

XRPD on Crystalline Compound 1 (Pattern 3)

The powder diffraction data were obtained with a Bruker D8 GADDSdiffractometer (Bruker-AXS, Karlsruhe, Germany) which was equipped witha monochromated CuKα source operating at a tube load of 40 kV and 40 mA.Powder samples were placed in sealed glass capillaries of 0.5 mm or 0.6mm in diameter; the capillary was rotated during data collection. Thesample-detector distance was 15 cm. Data were collected for 3≦2θ≦35°with a sample exposure time of at least 1200 seconds. The resultingtwo-dimensional diffraction arcs were integrated to create a traditional1-dimensional PXRD pattern with a step size of 0.02 degrees 2θ in therange of 3 to 35 degrees 2θ.

The X-Ray powder diffraction pattern for Pattern 3 of the free acid(Compound 1) is displayed in FIG. 13. Characteristic peaks include 5.5°2-Theta, 5.9° 2-Theta, 12.6° 2-Theta, 16.7° 2-Theta.

Single Crystal Data

Data were collected on a Bruker X8 APEX2 CCD diffractometer (Bruker AXS,Inc, Madison, Wis.). Intensities were measured using Cu Kα radiation(λ=1.5418 Å) at a constant temperature with φ and ω variable scantechnique and were corrected only for Lorentz-polarization factors.Indexing and processing of the measured intensity data were carried outwith the APEX2 software package/program suite. Alternately, singlecrystal data were collected on a Bruker-Nonius Kappa CCD 2000 systemusing Cu Kα radiation (λ=1.5418 Å). Indexing and processing of themeasured intensity data were carried out with the HKL2000 softwarepackage (Otwinowski, Z. & Minor, W. (1997) in MacromolecularCrystallography, eds. Carter, W. C. Jr & Sweet, R. M. (Academic, NY),Vol. 276, pp. 307-326) in the Collect program suite (Collect Datacollection and processing user interface: Collect: Data collectionsoftware, R. Hooft, Nonius B. V., 1998). When indicated, crystals werecooled in the liquid nitrogen cold stream of an Oxford cryosystem(Oxford Cryosystems Cryostream cooler: J. Cosier and A. M. Glazer, J.Appl. Cryst., 1986, 19, 105) during data collection.

The structures were solved by direct methods and refined on the basis ofobserved reflections using either the SDP (SDP, Structure DeterminationPackage, Enraf-Nonius, Bohemia N.Y. 11716) software package with minorlocal modifications or the crystallographic packages MAXUS (maXussolution and refinement software suite: S. Mackay, C. J. Gilmore. C.Edwards, M. Tremayne, N. Stewart, K. Shankland. maXus: a computerprogram for the solution and refinement of crystal structures fromdiffraction data) or SHELXTL (SHELXTL: Bruker-AXS, 5465 East CherylParkway, Madison, Wis., 53711, USA).

The derived atomic parameters (coordinates and temperature factors) wererefined through full matrix least-squares. The function minimized in therefinements was Σ_(W)(|F_(O)|−|F_(C)|)². R is defined as Σ∥F_(O)|−|F_(C)∥/Σ|F_(O)| whileR_(W)=[Σ_(W)(|F_(O)|−|F_(C)|)²/Σ_(W)|F_(O)|²]^(1/2) where w is anappropriate weighting function based on errors in the observedintensities. Difference maps were examined at all stages of refinement.Hydrogens were introduced in idealized positions with isotropictemperature factors, but no hydrogen parameters were varied.

The single crystal X-ray measurements for Compound 1 (Pattern 1) areshown in Table 1 and Table 2. For Compound 1 (Pattern 1), when singlecrystals are unstable at room temperature, X-ray diffraction experimentswould typically be conducted at a lower temperature to help stabilizethe crystal and to obtain diffraction data sufficient for the structuresolution and refinement. Low temperature data collection also has theadvantage of increasing signal/background ratios and thus improvesdiffraction intensities and the resolution in general. In this case, thecrystal structure was first solved with software program SHELXTL(Bruker-AXS, 2008, Madison, Wis.) using a complete dataset collected at203K. A short data collection of 20 minutes was then carried out at roomtemperature to determine the room temperature unit cell parameters viasoftware APEX2 (Bruker-AXS, 2010, Madison, Wis.). The crystal structureat room temperature was then generated by refining the atomiccoordinates using the LT intensity data and the unit cell parametersobtained at RT using software SHELXTL. A room temperature powder X-raypattern was calculated from the temperature-adjusted atomic coordinatesand unit cell parameters using software Lattice View, and the simulatedpattern matched the bulk PXRD collected at RT. This procedure can beapplied when there is no phase transition within the temperature rangeand the iso-structures are neat.

TABLE 1 Crystal Data of Compound 1 (Pattern 1) at 25° C. a(Å) 26.2070(8)b(Å) 37.700(1) c(Å) 5.0051(2) α ° 90 β° 90 γ ° 90 V(Å3) 4945.1(3) Z 8Calculated Density 1.296 Crystal System Orthorhombic SG P2₁2₁2 R1 0.0418Sol. Sites —

TABLE 2 Fractional Atomic Coordinates for Compound 1 (Pattern 1) at 25°C. Atom x y z O1 0.2368 0.8973 0.6933 O2 0.2386 0.8401 0.5701 H2A 0.26170.8474 0.4743 O3 0.0273 0.5978 0.4093 O4 0.1763 0.5542 0.1454 O5 0.22410.5349 0.4986 N1 0.0212 0.5620 0.3259 N2 0.1500 0.5615 0.5784 H2 0.15990.5632 0.7419 C1 0.2189 0.8662 0.6972 C2 0.1723 0.8581 0.8574 C3 0.16440.8804 1.1107 H3A 0.1478 0.8692 1.2617 H3B 0.1909 0.8973 1.1577 C40.1330 0.8881 0.8707 H4A 0.1403 0.9097 0.7723 H4B 0.0972 0.8816 0.8762C5 0.1542 0.8201 0.8423 C6 0.1176 0.8102 0.6565 H6 0.1012 0.8276 0.5560C7 0.1051 0.7749 0.6186 H7 0.0808 0.7692 0.4901 C8 0.1274 0.7476 0.7649C9 0.1621 0.7580 0.9599 H9 0.1769 0.7407 1.0675 C10 0.1752 0.7932 0.9992H10 0.1984 0.799 1.1326 C11 0.1146 0.7098 0.7087 C12 0.0793 0.70110.5088 H12 0.0639 0.7192 0.4122 C13 0.0665 0.6662 0.4502 H13 0.04250.6615 0.3178 C14 0.0889 0.6384 0.5860 C15 0.1243 0.6465 0.7876 H150.1396 0.6282 0.8831 C16 0.1367 0.6815 0.8462 H16 0.1604 0.6861 0.9802C17 0.0756 0.6020 0.5125 C18 0.0643 0.5464 0.3846 C19 0.0725 0.50770.3186 H19A 0.0414 0.4948 0.3492 H19B 0.0990 0.4982 0.4303 H19C 0.08220.5054 0.1345 C20 0.0991 0.5703 0.5047 C21 0.1831 0.5502 0.3847 C220.2633 0.5211 0.3136 H22 0.2716 0.5395 0.1823 C23 0.2440 0.4882 0.1668H23A 0.2164 0.4947 0.0508 H23B 0.2323 0.4710 0.2944 H23C 0.2713 0.47820.0629 C24 0.3100 0.5150 0.4952 C25 0.3297 0.5435 0.6389 H25 0.31480.5658 0.6209 C26 0.3710 0.5392 0.8083 H26 0.3839 0.5584 0.9034 C270.3932 0.5058 0.8349 H27 0.4209 0.5027 0.9487 C28 0.3743 0.4776 0.6936H28 0.3895 0.4554 0.7105 C29 0.3324 0.4818 0.5248 H29 0.3194 0.46240.4321 O11 0.3128 0.8572 0.2537 O12 0.3166 0.9142 0.3767 H12A 0.29260.9081 0.4708 O13 0.5345 0.6293 −0.3192 O14 0.3763 0.6325 −0.3003 O150.3344 0.6304 −0.6979 N11 0.5380 0.5933 −0.4024 N12 0.4180 0.6211−0.6935 H12B 0.4147 0.6179 −0.8627 C31 0.3350 0.8862 0.2512 C32 0.38400.8912 0.1076 C33 0.4171 0.9226 0.1868 H33A 0.4058 0.9370 0.3365 H33B0.4538 0.9199 0.1722 C34 0.3876 0.9250 −0.0628 H34A 0.4062 0.9236−0.2299 H34B 0.3583 0.9407 −0.0655 C35 0.4071 0.8572 0.0081 C36 0.45010.8421 0.1226 H36 0.4675 0.8542 0.2566 C37 0.4674 0.8090 0.0384 H370.4964 0.7995 0.1185 C38 0.4431 0.7896 −0.1606 C39 0.4003 0.8057 −0.2798H39 0.3832 0.7941 −0.4169 C40 0.3832 0.8386 −0.1964 H40 0.3549 0.8485−0.2795 C41 0.4586 0.7529 −0.2356 C42 0.5056 0.6984 −0.1486 H42 0.52920.6862 −0.045 C43 0.4948 0.7336 −0.0908 H43 0.5121 0.7447 0.0482 C440.4813 0.6813 −0.3607 C45 0.4465 0.7006 −0.5135 H45 0.4304 0.6899−0.6578 C46 0.4358 0.7355 −0.4529 H46 0.4127 0.7478 −0.5591 C47 0.49150.6438 −0.4254 C48 0.4975 0.5878 −0.5550 C49 0.4884 0.5527 −0.6848 H49A0.5124 0.5357 −0.6172 H49B 0.4926 0.5550 −0.8746 H49C 0.4543 0.5449−0.6461 C50 0.4669 0.6186 −0.5731 C51 0.3764 0.6286 −0.5416 C52 0.29030.6492 −0.5923 H52 0.2884 0.6459 −0.3984 C53 0.2435 0.6324 −0.7266 H53A0.2415 0.6078 −0.6790 H53B 0.2465 0.6346 −0.9170 H53C 0.2131 0.6444−0.6675 C54 0.2947 0.6885 −0.6562 C55 0.2676 0.7132 −0.5051 H55 0.24740.7053 −0.3642 C56 0.2700 0.7492 −0.5600 H56 0.2513 0.7653 −0.4585 C570.3005 0.7610 −0.7671 H57 0.3030 0.7851 −0.8036 C58 0.3273 0.7366−0.9201 H58 0.3475 0.7445 −1.0606 C59 0.3242 0.7008 −0.8664 H59 0.34220.6848 −0.9719

A simulated XRPD obtained from the single crystal data for Compound 1(Pattern 1) matched the experimental XRPD.

The single crystal X-ray measurements for Compound 1 (Pattern 2) areshown in Table 3 and Table 4.

TABLE 3 Crystal Data of Compound 1 (Pattern 2) at 25° C. a(Å) 30.3522(9)b(Å) 7.8514(3) c(Å) 22.4570(7) α ° 90 β° 111.665(2) γ ° 90 V(Å³)4973.6(3) Z 8 Calculated Density 1.289 Crystal System Monoclinic SG C2R1 0.0298 Sol. Sites —

TABLE 4 Fractional Atomic Coordinates for Compound 1 (Pattern 2) at 25°C. Atom x y z O1A 0.4270 0.2955 0.8458 O2A 0.3936 0.3803 0.7425 O3A0.2474 0.2796 0.6497 O4A 0.3113 −0.8496 0.4058 H4OA 0.2947 −0.91010.3763 O5A 0.3678 −0.9522 0.3764 N1A 0.3520 0.2317 0.7904 H1NA 0.35360.1616 0.8206 N2A 0.2455 0.4272 0.6847 C1A 0.4836 0.8847 0.8164 H1A0.486 0.9997 0.8081 C2A 0.4490 0.8319 0.8373 H2A 0.4285 0.9105 0.8442C3A 0.4447 0.6587 0.8481 H3A 0.4213 0.6220 0.8626 C4A 0.4748 0.54220.8377 C5A 0.5098 0.6023 0.8171 H5A 0.5306 0.5254 0.8097 C6A 0.51440.7727 0.8074 H6A 0.5386 0.8110 0.7948 C7A 0.4732 0.3557 0.8491 H7A0.4824 0.2940 0.8175 C8A 0.5065 0.3071 0.9156 H8A 0.5066 0.1855 0.9202H8B 0.4960 0.3592 0.9467 H8C 0.5379 0.3456 0.922 C9A 0.3915 0.30950.7889 C10A 0.3084 0.2691 0.7412 C11A 0.2818 0.4188 0.7385 C12A 0.29250.5557 0.7873 H12A 0.3185 0.6233 0.7859 H12B 0.3009 0.5058 0.8291 H12C0.2651 0.6266 0.7786 C13A 0.2863 0.1884 0.6849 C14A 0.2963 0.0390 0.6532C15A 0.3254 −0.0908 0.6873 H15A 0.3374 −0.0878 0.7319 C16A 0.3371−0.2254 0.6561 H16A 0.3561 −0.3131 0.6798 C17A 0.3205 −0.2305 0.5894C18A 0.2905 −0.1011 0.5557 H18A 0.2784 −0.1041 0.5112 C19A 0.2783 0.03140.5864 H19A 0.2580 0.1161 0.5627 C20A 0.3360 −0.3652 0.5546 C21A 0.3417−0.5332 0.5743 H21A 0.335 −0.565 0.6100 C22A 0.3570 −0.6542 0.5419 H22A0.3599 −0.7669 0.5556 C23A 0.3682 −0.6111 0.4896 C24A 0.3622 −0.44410.4694 H24A 0.369 −0.4130 0.4338 C25A 0.3462 −0.3214 0.5010 H25A 0.3422−0.2096 0.4863 C26A 0.3879 −0.7421 0.4571 C27A 0.4357 −0.8181 0.4939H27A 0.4517 −0.7817 0.5380 H27B 0.4407 −0.9366 0.4859 C28A 0.4317−0.6961 0.4438 H28A 0.4341 −0.7387 0.4046 H28B 0.4452 −0.5838 0.4567C29A 0.3555 −0.8585 0.4099 O1B 0.4377 −0.2507 0.3549 O2B 0.4102 −0.41370.2659 O3B 0.2606 −0.3039 0.1508 O4B 0.3053 0.8448 −0.0891 H4OB 0.28910.8813 −0.1247 O5B 0.3592 1.0126 −0.1017 N1B 0.3614 −0.2468 0.2959 H1NB0.3593 −0.1765 0.3241 N2B 0.2589 −0.4546 0.1844 C1B 0.5329 0.2266 0.3192H1B 0.5434 0.3320 0.3108 C2B 0.4877 0.2084 0.3179 H2B 0.4672 0.30140.3082 C3B 0.4722 0.0515 0.3311 H3B 0.4415 0.0405 0.3301 C4B 0.5017−0.0872 0.3456 C5B 0.5478 −0.0661 0.3465 H5B 0.5685 −0.1581 0.3565 C6B0.5625 0.0896 0.3329 H6B 0.5930 0.1015 0.333 C7B 0.4864 −0.2622 0.3584H7B 0.4880 −0.3413 0.3255 C8B 0.5159 −0.3299 0.4230 H8D 0.5070 −0.44550.4267 H8E 0.5109 −0.2617 0.4554 H8F 0.5488 −0.3258 0.4284 C9B 0.4038−0.3123 0.3020 C10B 0.3201 −0.2928 0.2434 C11B 0.2947 −0.4451 0.2392C12B 0.3045 −0.5818 0.2877 H12D 0.2986 −0.5405 0.3243 H12E 0.3371−0.6162 0.3007 H12F 0.2844 −0.6776 0.2697 C13B 0.2984 −0.2109 0.1873C14B 0.3079 −0.0562 0.1578 C15B 0.3415 0.0597 0.1925 H15B 0.3572 0.04270.2363 C16B 0.3522 0.2009 0.1639 H16B 0.3753 0.2767 0.1886 C17B 0.32930.2316 0.0989 C18B 0.2950 0.1181 0.0647 H18B 0.2787 0.1369 0.0212 C19B0.2838 −0.0235 0.0929 H19B 0.2601 −0.0973 0.0684 C20B 0.3416 0.38370.0688 C21B 0.3521 0.5360 0.1009 H21B 0.351 0.5437 0.1417 C22B 0.36430.6776 0.0743 H22B 0.3708 0.7792 0.0972 C23B 0.3672 0.6726 0.0147 C24B0.3570 0.5200 −0.0175 H24B 0.3590 0.5123 −0.0577 C25B 0.3438 0.37730.0083 H25B 0.3364 0.2766 −0.0151 C26B 0.3823 0.8252 −0.0134 C27B 0.41470.9571 0.0303 H27C 0.4101 1.0750 0.0165 H27D 0.4243 0.9405 0.0761 C28B0.4342 0.8400 −0.0049 H28C 0.4557 0.7516 0.0194 H28D 0.4415 0.8862−0.0402 C29B 0.3486 0.9014 −0.0728

A simulated XRPD obtained from the single crystal data for Compound 1(Pattern 2) is displayed in FIG. 12.

The single crystal X-ray measurements for Compound 1 (Pattern 3) areshown in Table 5 and Table 6.

TABLE 5 Crystal Data of Compound 1 (Pattern 3) at 25° C. a(Å) 32.3574(9)b(Å) 5.1057(2) c(Å) 33.148(1) α ° 90 β° 114.846(2) γ ° 90 V(Å³)4969.4(3) Z 8 Calculated Density 1.290 Crystal System Monoclinic SG C2R1 0.0553 Sol. Sites —

TABLE 6 Fractional Atomic Coordinates for Compound 1 (Pattern 3) at 25°C. Atom x y z O1 0.1645 1.6687 0.7626 O2 0.1196 1.8240 0.6962 H2A 0.11051.9124 0.7115 O3 0.4706 0.3023 0.8618 O4 0.4383 0.5185 0.9547 O5 0.38610.1933 0.9771 N1 0.5029 0.1165 0.8877 N2 0.4213 0.0950 0.9346 H2 0.4133−0.0632 0.9369 C1 0.1537 1.6790 0.7220 C2 0.1783 1.5407 0.7005 C3 0.18171.6969 0.6629 H3A 0.1686 1.8713 0.6573 H3B 0.2093 1.6782 0.6584 C40.1515 1.4832 0.6515 H4A 0.1598 1.3284 0.6397 H4B 0.1191 1.5215 0.6386C5 0.2169 1.3620 0.7273 C6 0.2519 1.3129 0.7160 H6 0.2525 1.4003 0.6916C7 0.2863 1.1405 0.7387 H7 0.3091 1.1133 0.7291 C8 0.2881 1.0073 0.7753C9 0.2529 1.0501 0.7870 H9 0.2526 0.9599 0.8113 C10 0.2179 1.2214 0.7644H10 0.1947 1.2446 0.7736 C11 0.3260 0.8265 0.8007 C12 0.3678 0.84170.7985 H12 0.3719 0.9683 0.7804 C13 0.4037 0.6776 0.8222 H13 0.43110.6947 0.8197 C14 0.3989 0.4867 0.8497 C15 0.3570 0.4654 0.8518 H150.3527 0.3365 0.8695 C16 0.3216 0.6341 0.8279 H16 0.2940 0.6173 0.8302C17 0.4360 0.3115 0.8747 C18 0.4879 0.0253 0.9154 C19 0.5138 −0.18230.9479 H19A 0.5361 −0.2572 0.9393 H19B 0.4932 −0.3164 0.9482 H19C 0.5287−0.1069 0.9770 C20 0.4461 0.1449 0.9092 C21 0.4095 0.2913 0.9558 C220.3667 0.3783 0.9973 H22 0.3847 0.5400 1.0041 C23 0.3719 0.2503 1.0408H23A 0.4030 0.1984 1.0574 H23B 0.3526 0.0989 1.0344 H23C 0.3634 0.37291.0579 C24A 0.3174 0.4369 0.9600 C25A 0.2892 0.2301 0.9360 H25A 0.29940.0587 0.943 C26A 0.2468 0.2743 0.9024 H26A 0.2288 0.1355 0.8864 C27A0.2314 0.5309 0.8929 H27A 0.2026 0.5649 0.8706 C28A 0.2590 0.7367 0.9167H28A 0.2487 0.9084 0.9103 C29A 0.3020 0.6860 0.9500 H29A 0.3205 0.82460.9656 C24B 0.3186 0.4368 0.9689 C25B 0.2986 0.4076 0.9229 H25B 0.31560.3433 0.9084 C26B 0.2532 0.4745 0.8987 H26B 0.2398 0.4550 0.8679 C27B0.2277 0.5706 0.9204 H27B 0.1973 0.6153 0.9041 C28B 0.2477 0.5997 0.9663H28B 0.2307 0.6640 0.9808 C29B 0.2932 0.5329 0.9906 H29B 0.3066 0.55241.0213 O11 0.5855 −0.3790 0.7389 O12 0.6296 −0.5381 0.8053 H12A 0.6393−0.6256 0.7904 O13 0.2850 1.0407 0.6414 O14 0.3349 1.5830 0.5527 O150.3640 1.2869 0.5212 N3 0.2519 1.2154 0.6127 N4 0.3300 1.1471 0.5621 H40.3369 0.9966 0.5549 C31 0.5959 −0.3928 0.7792 C32 0.5705 −0.2559 0.8006C33 0.5664 −0.4182 0.8373 H33A 0.5385 −0.4026 0.8413 H33B 0.5797 −0.59220.8426 C34 0.5967 −0.2012 0.8499 H34A 0.6291 −0.2382 0.8632 H34B 0.5880−0.0487 0.8619 C35 0.5326 −0.0721 0.7736 C36 0.4979 −0.0179 0.7855 H360.497 −0.1041 0.8099 C37 0.4639 0.1609 0.7626 H37 0.4414 0.1940 0.7724C38 0.4626 0.2888 0.7263 C39 0.4972 0.2384 0.7136 H39 0.4975 0.32440.6890 C40 0.5316 0.0621 0.7366 H40 0.5546 0.0328 0.7273 C41 0.42510.4749 0.7008 C42 0.3839 0.4726 0.7041 H42 0.3795 0.3514 0.7230 C430.3493 0.6423 0.6808 H43 0.3221 0.6343 0.684 C44 0.3543 0.8260 0.6524C45 0.3958 0.8349 0.6494 H45 0.4003 0.9590 0.6311 C46 0.4304 0.66260.6730 H46 0.4578 0.6725 0.6703 C47 0.3177 1.0047 0.6270 C48 0.26561.2775 0.5823 C49 0.2379 1.4597 0.5459 H49A 0.2094 1.4936 0.5475 H49B0.2322 1.3818 0.5177 H49C 0.2541 1.6212 0.5490 C50 0.3070 1.1500 0.5896C51 0.3423 1.3576 0.5461 C52 0.3836 1.4958 0.5041 H52 0.3667 1.65770.5027 C53 0.3743 1.4176 0.4576 H53A 0.3424 1.3812 0.4413 H53B 0.39161.2637 0.4582 H53C 0.3829 1.5576 0.4434 C54A 0.4328 1.5290 0.5409 C55A0.4615 1.3163 0.5609 H55A 0.4510 1.1470 0.552 C56A 0.5049 1.3533 0.5933H56A 0.5235 1.2105 0.6066 C57A 0.5205 1.6071 0.6059 H57A 0.5499 1.63510.6275 C58A 0.4923 1.8191 0.5861 H58A 0.5028 1.9889 0.5945 C59A 0.44841.7772 0.5537 H59A 0.4296 1.9195 0.5407 C54B 0.4328 1.5319 0.5311 C55B0.4607 1.5550 0.5089 H55B 0.4484 1.5408 0.4781 C56B 0.5071 1.5994 0.5328H56B 0.5258 1.6148 0.5180 C57B 0.5256 1.6207 0.5788 H57B 0.5566 1.65040.5948 C58B 0.4976 1.5976 0.6010 H58B 0.5099 1.6119 0.6318 C59B 0.45121.5532 0.5772 H59B 0.4325 1.5378 0.5920

A simulated XRPD obtained from the single crystal data for Compound 1(Pattern 3) is displayed in FIG. 13.

The single crystal X-ray measurements for Compound 2 (Pattern 1) areshown in Table 7 and Table 8.

TABLE 7 Crystal Data of Compound 2 (Pattern 1) at 25° C. a(Å) 13.8714(2)b(Å) 7.7379(2) c(Å) 25.5253(5) α ° 90 β° 103.863(1) γ ° 90 V(Å3)2659.96(9) Z 4 Calculated Density 1.305 Crystal System Monoclinic SG P2₁R1 0.0301 Sol. Sites 1H₂O

TABLE 8 Fractional Atomic Coordinates for Compound 2 (Pattern 1) at 25°C. Atom x y z Na1 0.5534 0.5155 0.7376 Na2 0.4268 0.1578 0.7661 O10.5791 0.6272 0.8275 O2 0.4955 0.3834 0.8159 O3 0.9547 0.9797 0.8944 O40.7250 0.4863 0.7547 O5 0.8692 0.6302 0.7635 O11 0.4860 0.2928 0.6884O12 0.4095 0.0426 0.6759 O13 −0.0533 −0.8204 0.4086 O14 −0.2536 −0.35960.2463 O15 −0.1150 −0.5250 0.2643 N1 0.8774 1.0896 0.8666 N2 0.77990.6684 0.8247 H2A 0.7369 0.6305 0.8411 N11 −0.1310 −0.9306 0.3820 N12−0.2104 −0.5182 0.3228 H12A −0.2539 −0.4666 0.3362 C1 0.5492 0.48980.8454 C2 0.5797 0.4598 0.9052 C3 0.4988 0.3880 0.9302 H3A 0.4986 0.42450.9665 H3B 0.4336 0.3698 0.9066 C4 0.5795 0.2744 0.9234 H4A 0.5637 0.1860.8957 H4B 0.6287 0.2407 0.9556 C5 1.3459 0.0837 1.0622 C6 1.3747 0.24121.0465 H6 1.4415 0.2714 1.0560 C7 1.3071 0.3555 1.0170 H7 1.3291 0.46141.0072 C8 1.2061 0.3160 1.0015 C9 1.1770 0.1582 1.0172 H9 1.1102 0.12771.0076 C10 1.2455 0.0434 1.0473 H10 1.2237 −0.0621 1.0575 C11 1.13330.4415 0.9706 C12 1.1456 0.6172 0.9799 H12 1.1993 0.6559 1.0066 C131.0812 0.7356 0.9508 H13 1.0925 0.8527 0.9579 C14 0.9995 0.6851 0.9111C15 0.9854 0.5090 0.9018 H15 0.9309 0.4708 0.8755 C16 1.0508 0.38970.9310 H16 1.0396 0.2725 0.9240 C17 0.9295 0.8131 0.8832 C18 0.80970.9856 0.8392 C19 0.7147 1.0541 0.8044 H19A 0.7093 1.1752 0.8113 H19B0.6596 0.9937 0.8124 H19C 0.7146 1.0373 0.7671 C20 0.8390 0.8108 0.8477C21 0.7864 0.5881 0.7789 C22 0.8734 0.5694 0.7100 H22 0.8543 0.44730.7061 C23 0.9804 0.5886 0.7073 H23A 1.0223 0.5267 0.7367 H23B 0.99830.7087 0.7098 H23C 0.9887 0.5425 0.6738 C24 0.8038 0.6735 0.6678 C250.8000 0.8512 0.6713 H25 0.8405 0.9078 0.7006 C26 0.7367 0.9449 0.6320H26 0.7344 1.0646 0.6348 C27 0.6772 0.8642 0.5888 H27 0.6343 0.92870.5624 C28 0.6801 0.6930 0.5843 H28 0.6395 0.6381 0.5546 C29 0.74380.5963 0.6238 H29 0.7455 0.4768 0.6202 C31 0.4410 0.1780 0.6584 C320.4204 0.2015 0.5979 C33 0.4254 0.3816 0.5778 H33A 0.3815 0.4114 0.5433H33B 0.4374 0.4742 0.6041 C34 0.5076 0.2623 0.5774 H34A 0.5699 0.28140.6037 H34B 0.5140 0.2186 0.5428 C35 0.3483 0.0785 0.5641 C36 0.3786−0.0804 0.5489 H36 0.4453 −0.1107 0.5597 C37 0.3122 −0.1948 0.5183 H370.3350 −0.3003 0.5087 C38 0.1813 0.0035 0.5165 H38 0.1147 0.0343 0.5057C39 0.2112 −0.1555 0.5014 C40 0.2480 0.1178 0.5473 H40 0.2252 0.22320.5571 C41 0.1393 −0.2825 0.4702 C42 0.1502 −0.4561 0.4816 H42 0.2045−0.4932 0.5081 C43 0.0831 −0.5762 0.4551 H43 0.0927 −0.6926 0.4640 C440.0018 −0.5265 0.4155 C45 −0.0105 −0.3529 0.4038 H45 −0.0648 −0.3160.3772 C46 0.0569 −0.2331 0.4310 H46 0.0465 −0.1165 0.4228 C47 −0.0718−0.6564 0.3900 C48 −0.1921 −0.8294 0.3485 C49 −0.2850 −0.9024 0.3122H49A −0.2753 −1.0228 0.3059 H49B −0.2995 −0.8416 0.2784 H49C −0.3394−0.8894 0.3291 C50 −0.1585 −0.6569 0.3520 C51 −0.1972 −0.4595 0.2751 C52−0.0952 −0.4900 0.2111 H52 −0.0361 −0.5577 0.2100 C53 −0.0661 −0.30360.2074 H53A −0.0165 −0.2728 0.2392 H53B −0.1234 −0.2313 0.2045 H53C−0.0400 −0.2880 0.1762 C54 −0.1780 −0.5628 0.1670 C55 −0.2312 −0.46370.1244 H55 −0.2167 −0.347 0.1222 C56 −0.3051 −0.5386 0.0854 H56 −0.3410−0.4712 0.0572 C57 −0.3266 −0.7096 0.0874 H57 −0.3763 −0.7582 0.0603 C58−0.2751 −0.8119 0.1294 H58 −0.2896 −0.9287 0.1315 C59 −0.2012 −0.73300.1681 H59 −0.1655 −0.8002 0.1964 O21 0.5365 0.7582 0.6857 H21A 0.57400.7620 0.6607 H21B 0.5040 0.8610 0.6889 O22 0.4576 −0.0839 0.8173 H22A0.4060 −0.1190 0.8329 H22B 0.5040 −0.1760 0.8212

A simulated XRPD obtained from the single crystal data for Compound 2(Pattern 1) matched the experimental XRPD.

Example 13: Differential Scanning Calorimetry (DSC) andThermogravimetric Analysis (TGA)

DSC data were collected on a TA Instruments Q2000 equipped with a 50position auto-sampler. The calibration for thermal capacity was carriedout using sapphire and the calibration for energy and temperature wascarried out using certified indium. Typically 0.5-3 mg of each sample,in a pin-holed aluminum pan, was heated at 10° C.·min⁻¹ from 25° C. to250° C. A purge of dry nitrogen at 50 ml min⁻¹ was maintained over thesample. The instrument control software was Advantage for Q Seriesv2.8.0.392 and Thermal Advantage v4.8.3 and the data were analyzed usingUniversal Analysis v4.3A.

TGA data were collected on a TA Instruments Q500 TGA, equipped with a 16position auto-sampler. The instrument was temperature calibrated usingcertified Alumel. Typically 5-30 mg of each sample was loaded onto apre-tared platinum crucible and aluminum DSC pan, and was heated at 10°C.·min from ambient temperature to 350° C. A nitrogen purge at 60ml·min⁻¹ was maintained over the sample. The instrument control softwarewas Advantage for Q Series v2.8.0.392 and Thermal Advantage v4.8.3.

Pattern 1 Free Acid (Compound 1)

A sample of the free acid (Compound 1) were analyzed by TGA and DSC andthe thermograms are shown in FIG. 2 and FIG. 3. No mass loss is noted inthe TGA up to 150° C. and a sharp endotherm, attributed to a melt, isobserved at around onset 172° C.-176° C. in the DSC.

Amorphous Compound 2

A sample of the amorphous sodium salt acid (Compound 2) were analyzed byTGA and DSC. A 2.79% mass loss is observed in the TGA up to 150° C.,likely attributable to loss of ethanol and water. A DSC thermogram isshown in FIG. 11.

Crystalline Compound 2 (Pattern 1)

A sample of the hydrated crystalline sodium salt (Compound 2) wereanalyzed by TGA and DSC and the thermograms are shown in FIG. 5 and FIG.6.

Example 14: Water Determination by Karl Fischer Titration (KF)

The water content of each sample was measured on a Mettler Toledo DL39Coulometer using Hydranal Coulomat AG reagent and an argon purge.Weighed solid samples were introduced into the vessel on a platinum TGApan which was connected to a subaseal to avoid water ingress. Approx 10mg of sample was used per titration and duplicate determinations weremade.

The water content of the crystalline Compound 2 (Pattern 1) wasdetermined as 4.1% m/m by Karl Fisher titration, which correlates withthe mass loss observed in the TGA and calculates as 1.2 mol of water permol of API. All the data is consistent with crystalline Compound 2(Pattern 1) being a hydrated crystalline form.

Example 15: Gravimetric Vapour Sorption (GVS)

In some embodiments, sorption isotherms were obtained using a SMS DVSIntrinsic moisture sorption analyzer, controlled by SMS Analysis Suitesoftware. The sample temperature was maintained at 25° C. by theinstrument controls. The humidity was controlled by mixing streams ofdry and wet nitrogen, with a total flow rate of 200 ml·min−1. Therelative humidity was measured by a calibrated Rotronic probe (dynamicrange of 1.0-100% RH), located near the sample. The weight change (massrelaxation) of the sample as a function of % RH was constantly monitoredby the microbalance (accuracy ±0.005 mg). Typically 5-20 mg of samplewas placed in a tared mesh stainless steel basket under ambientconditions. The sample was loaded and unloaded at 40% RH and 25° C.(typical room conditions). A moisture sorption isotherm was performed asoutlined below (2 scans giving 1 complete cycle). The standard isothermwas performed at 25° C. at 10% RH intervals over a 0.5-90% RH range.

TABLE 9 Method Parameters for SMS DVS Intrinsic Experiments ParametersValues Adsorption - Scan 1 40-90 Desorption/Adsorption - Scan 2 85-Dry,Dry-40 Intervals (% RH) 10 Number of Scans 2 Flow rate (ml · min−1) 200Temperature (° C.) 25 Stability (° C. · min−1) 0.2 Sorption Time (hours)6 hour time out

The sample was recovered after completion of the isotherm andre-analyzed by XRPD.

Crystalline Compound 1 (Pattern 1)

The free acid (Compound 1) is less hygroscopic than the sodium salt,with only a 0.45% mass gain between 0% and 90% relative humidity. TheXRPD of the crystalline free acid was substantially the same afterstorage at 40° C./75% RH for one week.

Amorphous Compound 2

Amorphous Compound 2 exhibits hygroscopicity, increasing in mass by morethan 20% upon raising the relative humidity from 0% to 90%. No change ofform is observed during this process.

The material remains largely amorphous in nature after storage at 40°C./75% RH for one week.

Crystalline Compound 2 (Pattern 1)

Crystalline Compound 2 (Pattern 1) exhibits lower hygroscopicity thanits amorphous counterpart and shows no loss of crystallinity after aweek's storage at 40° C./75% RH or 25° C./95% RH.

Example 16: Thermodynamic Aqueous Solubility

Aqueous solubility was determined by suspending sufficient compound inwater to give a maximum final concentration of ≧10 mg·ml−1 of the parentfree-form of the compound. The suspension was equilibrated at 25° C. for24 hours then the pH was measured. The suspension was then filteredthrough a glass fiber C filter into a 96 well plate unless statedotherwise. The filtrate was then diluted by a factor of 101.Quantification was by HPLC with reference to a standard solution ofapproximately 0.25 mg·ml⁻¹ in DMSO. Different volumes of the standard,diluted and undiluted sample solutions were injected. The solubility wascalculated using the peak areas determined by integration of the peakfound at the same retention time as the principal peak in the standardinjection.

For solubility assessment at various pH levels sufficient material toproduce a maximum concentration of 10 mg·ml⁻¹ of API was treated with0.15M NaCl solution and then the pH adjusted with HCl or NaOH solutionsto achieve the desired pH levels. The suspensions were allowed toequilibrate for 2 hours and the pH measured and adjusted if necessary.Suspensions were then filtered and the amount of dissolved APIquantified by HPLC against a standard reference solution.

TABLE 18 HPLC Method Parameters for Solubility Measurements Type ofmethod: Reverse phase with gradient elution Column: Phenomenex Luna,C18(2) 5 μm 50 × 4.6 mm Column Temperature (° C.): 25 StandardInjections (μl): 1, 2, 3, 5, 7, 10 Test Injections (μl): 1, 2, 3, 10,20, 50 Detection: 260, 80 Wavelength, Bandwidth (nm): Flow Rate (ml ·min⁻¹): 2 Phase A: 0.1% TFA in water Phase B: 0.085% TFA in acetonitrileTimetable: Time (min) % Phase A % Phase B 0.0 95 5 1.0 80 20 2.3 5 953.3 5 95 3.5 95 5 4.4 95 5

Analysis was performed on an Agilent HP1100 series system equipped witha diode array detector and using ChemStation software vB.02.01-SR1.

The thermodynamic aqueous solubility of both the free acid (Compound 1)and the sodium salt (Compound 2) were determined.

TABLE 11 Thermodynamic aqueous solubility pH of Solubility saturated(mg/ml, free Form Solvent solution Appearance form equivalent) free acidwater 7.07 Suspension 0.028 free acid water 6.93 Suspension 0.031 sodiumsalt water 8.90 Clear Solution >=20 sodium salt water 9.07 ClearSolution >=10

The solubility of both the free acid and amorphous sodium salt wasdetermined at a range of pH values. The results are shown in Tables 14and 15.

TABLE 12 Solubility profile for Compound 2 (Amorphous) pH pH after afterSolu- Target Appearance 2 Appearance 24 bility pH after 2 hrs hrs after24 hrs hrs (mg/mL) pH 3 Residual Solid 4.04 Residual Solid 2.90 <0.001pH 4 Residual Solid 4.29 Residual Solid 4.28 <0.001 pH 5 Residual Solid5.34 Residual Solid 5.11 <0.001 pH 6 Residual Solid 6.42 Residual Solid6.23 0.0017 pH 7 Suspension 7.18 Suspension 7.17 0.3 pH 8 FineSuspension 8.08 Fine Suspension 8.01 5.6 pH 9 Clear Solution 9.09 ClearSolution 9.07 17 pH 11 Clear Solution 11.07 Clear Solution 11.01 16

TABLE 13 Solubility profile for Compound 1 pH pH after after Solu-Target Appearance 2 Appearance 24 bility pH after 2 hrs hrs after 24 hrshrs (mg/mL) pH 3 Residual Solid/ 3.04 Residual Solid/ 3.04 <0.001 Solidon Surface Solid on Surface pH 4 Residual Solid/ 3.88 Residual Solid/3.86 <0.001 Solid on Surface Solid on Surface pH 5 Residual Solid/ 4.81Residual Solid/ 4.81 <0.001 Solid on Surface Solid on Surface pH 6Residual Solid/ 5.83 Residual Solid/ 5.82 <0.001 Solid on Surface Solidon Surface pH 7 Residual Solid/ 6.92 Suspension/ 6.85 0.0033 Solid onSurface Residual Solid pH 8 Suspension 7.95 Suspension 7.90 0.035 pH 9Suspension 8.92 Suspension 8.90 0.93 pH 11 Suspension 8.84 ClearSolution 11.05 13

Example 17. Chemical Purity Determination

Purity analysis was performed by HPLC on an Agilent HP1100 series systemequipped with a diode array detector and using ChemStation softwarevB.02.01-SR1.

TABLE 14 HPLC Method Parameters for Chemical Purity DeterminationsSample Preparation: ~0.5 mg/ml in acetonitrile:water 1:1 v/v Column:Supelco Ascentis Express C18, 100 × 4.6 mm, 2.7 μm Column Temperature (°C.): 25 Injection (μl): 2-5 Detection: Wavelength, Bandwidth (nm): 255,90 Flow Rate (ml · min⁻¹):   2.0 Phase A: 0.1% TFA in water Phase B:0.085% TFA in acetonitrile Time (min) % Phase A % Phase B Timetable: 095 5 6 5 95 6.2 95 5 8 95 5

Samples of Compound 1 and Compound 2 were found to be greater than 90%pure. In some embodiments, samples of Compound 1 were found to begreater than 95% pure, greater than 96% pure, greater than 97% pure,greater than 98% pure, greater than 99% pure. In some embodiments,samples of Compound 2 were found to be greater than 94% pure, greaterthan 95% pure, greater than 96% pure, greater than 97% pure, greaterthan 98% pure, greater than 99% pure.

In some embodiments, samples of Compound 1 or Compound 2 include adetectable amount of a compound having one of the following structures:

or combinations thereof.

Example 18. Chiral Purity

Chiral purity analysis was performed by HPLC on an Agilent HP1100 seriessystem equipped with a diode array detector and using ChemStationsoftware vB.02.01-SR1.

TABLE 15 HPLC Method Parameters for Chemical Purity DeterminationsSample Preparation: ~0.5 mg/ml in methanol Column: Chiralpak AD-H, 5 μm,250 × 4.6 mm Column Temperature (° C.): 35 Injection (μl): 5 Detection:Wavelength, Bandwidth(nm): 290 Flow Rate (ml · min⁻¹): 1.0 Phase A:Hexane (0.05% TFA) Phase B: Isopropanol Time (min) % Phase A % Phase BTimetable:  0 80 20 35 80 20

Chiral purity (% enatiomeric excess: % e.e.) was determined. In someembodiments, samples of Compound 1 and Compound 2 were found to have achiral purity greater than 98%. In some embodiments, samples of Compound1 were found to have a chiral purity greater than 95%, greater than 96%,greater than 97%, greater than 98%, greater than 99%. In someembodiments, samples of Compound 2 were found to have a chiral puritygreater than 94%, greater than 95%, greater than 96%, greater than 97%,greater than 98%, greater than 99%.

Example 19: Heavy Metals (Pd) by ICP-AES

Trace palladium (Pd) resulting from the use of catalytic amounts of Pdin the synthesis is assayed by inductively coupled plasma atomicemission spectrometry (ICP-AES). Pd content by ICP-AES is a detectableamount of palladium that is less than about 20 ppm. Pd content byICP-AES is less than about 20 ppm. Pd content by ICP-AES is a detectableamount of palladium that is less than 20 ppm, less than 15 ppm, lessthan 10 ppm, or less than 5 ppm. Pd content by ICP-AES is less than 20ppm, less than 15 ppm, less than 10 ppm, or less than 5 ppm. In someembodiments, samples or pharmaceutical compositions do not include adetectable amount of palladium.

Example 20: Heavy Metals (as Lead)

This test is performed according to USP<231>Method II.

Example 21: IR Spectroscopy of Crystalline Compound 2 (Pattern 1)

A sample of crystalline Compound 2 (Pattern 1; hydrate) was analyzed byinfrared spectroscopy. The data was collected on a Perkin-Elmer SpectrumOne instrument fitted with a universal Attenuated Total Reflectance(ATR) sampling accessory. Scan range=4000 cm-1 to 600 cm-1 with 64 scansand a resolution of 4 cm⁻¹. The data was analysed using Spectrum v5.0.1software.

Wavenumber (cm−¹) Intensity (% T) 3627 93 3345 94 3078 94 1707 80 163693 1574 75 1436 91 1376 78 1350 85 1325 85 1299 86 1194 91 1087 89 105989 1029 87 951 90 884 91 823 85 791 87 773 85 762 85 741 89 699 76 66591

Pharmaceutical Compositions

Pharmaceutical compositions that include Compound 1, includingpharmaceutically acceptable salts (e.g. Compound 2) and/orpharmaceutically acceptable solvates thereof include a variety of forms.In one aspect, pharmaceutical compositions are in the form of oraldosage forms. In some embodiments, pharmaceutical compositions areformulated as: oral solutions, oral suspensions, tablets, pills,capsules, ointments, creams or gels.

Example 22: Oral Solutions

In one aspect, an oral pharmaceutical composition in the form of an oralsolution is prepared as outlined below.

An oral solution is prepared at 50 mg/mL of Compound 1 or Compound 2.

Oral Solution A:

In one embodiment, an oral pharmaceutical composition is prepared withthe following ingredients:

-   -   50 mg/mL of Compound 1 or Compound 2    -   0.5% Methocel    -   0.5% Cherry flavor    -   0.5% sucralose    -   water, qs to

The manufacturing process for the oral solutions of Compound 1 orCompound 2 described above is as follows: weigh the required amount ofmethocel and transfer to the container. Add the required amount of waterto make a 0.5% solution and mix until dissolved. Weigh the requiredamount of cherry flavor and sucralose and add this to the solution andmix until homogenous. Weigh the required amount of Compound 1 orCompound 2 and slowly add to the solution. Mix until all Compound 1 orCompound 2 is dissolved (sonicate, warm, or stir if necessary).

Example 23: Capsule Formulations

Immediate Release Capsules

In one embodiment, a capsule formulation of Compound 1 or Compound 2 foradministration to humans is prepared with the following ingredients:

Quantity per Quantity per Size 4 Capsule Size 1 Capsule ComponentFunction mg mg Compound 1 or Active 10 to 500 mg 100 to 1000 mg Compound2 Hypromellose, USP Capsule Shell 1 capsule 1 capsule

The process to prepare Compound 1 or Compound 2 in a capsule is asfollows: Weigh the required amount of Compound 1 or Compound 2, add intothe appropriate size capsule, and close capsule. For example, 10-500 mgof Compound 1 or Compound 2 is placed into a Size 4 Capsule. In oneembodiment, 100-500 mg of Compound 1 or Compound 2 is placed into a Size1 Capsule.

Example 24: Immediate Release Tablets

Non-limiting examples of immediate release tables that include Compound2 are presented below.

TABLE 16 Immediate release Tablets Tablet 1 Tablet 2 Ingredients % w/wmg/unit % w/w mg/unit Compound 2 35.05 262.85 7.07 53.0 AnhydrousDibasic Calcium 19.98 149.85 29.31 219.8 Phosphate SilicifiedMicrocrystalline 39.96 299.70 58.62 439.7 Cellulose Sodium StarchGlycolate 3.00 22.50 3.00 22.50 Sodium Lauryl Sulfate 1.00 7.50 1.007.50 Magnesium Stearate 1.00 3.75 1.00 7.5 Total 100.00 750.00 100.00750.00

A non-limiting example for the preparation of immediate release tabletsis described below. Other dose amounts are contemplated. In some cases,the tablets are coated with a thin film (e.g. opadry coating).

Manufacturing/analytical equipment typically used in the preparation oftablets include: formulation (U.S.A. standard testing sieve: V-shellblender: ERWEKA TBH300 MD hardness tester; Vanderkamp friability tester;Manesty beta press, sixteen station); analytical (Agilent 1100 seriesHPLC with variable wavelength detector; VanKel model VK7000 dissolutionapparatus; VanKel model VK8000 dissolution autosampler).

To an appropriately-sized mixing vessel was add the following: ½ of thesilicified microcrystalline cellulose HD90, ½ of the anhydrous dibasicCalcium phosphate, Compound 2, sodium lauryl sulfate, sodium starchglycolate (intra-granular portion), remaining ½ of the anhydrous dibasiccalcium phosphate, remaining ½ of the silicified microcrystallinecellulose HD90. Blend ingredients. Pass blend through a sieve screen andtransfer back to mixing vessel. Blend ingredients. Pre-screen ½ ofmagnesium stearate (intra-granular amount) through a sieve screen. Addto powder blend and blend ingredients. Perform roller compaction of theblend using a roller compactor and appropriate parameters to generateribbons with suitable mechanical properties. Recycle the bypass (i.e.powder and loosely-compacted powder) through the roller compactor toachieve further densification. Pass the ribbons through a mill toachieve a granulation of suitable particle size distribution fortabletting. Transfer granulation to suitable mixing vessel. Blendgranulation. Pre-screen ½ of magnesium stearate (extra-granular amount)through a sieve screen. Add to granulation and blend ingredients.

Transfer the final granulation to a tablet press and compress intotablets.

Dissolution Studies

In some embodiments, all tablets are tested for dissolution using thefollowing parameters:

Dissolution Parameters Apparatus: USP2 Paddles Speed: 60 rpm DissolutionMedia: KH₂PO₄ buffer,, pH 7.4, 2% CTAB Dissolution volume: 900 mL Mediumtemperature: 37 ± 0.5° C. Sampling volume: 1.5 mL

Immediate release tablets show release no less than (NLT) 75% ofCompound 1 or Compound 2 within 45 minutes.

In some embodiments, tablets are optionally packaged in HDPE bottles,with CRC caps and heat induction seal.

Example 25: Enteric Coated Tablets

In some embodiments, enteric coated tablets are prepared with theingredients listed in Table 17.

TABLE 17 Enteric Coated Tablets Tablet # 1 Tablet #2 Tablet #3Ingredient Amount per Tablet (mg) Compound 1 or Compound 2 750 750 750immediate release tablet (750 mg) Eudragit L 100-55 20 Eudragit S 100 20Eudragit L 100 20 Triethyl Citrate 5 5 5 Acetone Purified Water Total775 775 775

The preparation of the enteric coated tablets is as follows: Weigh 388.0g of acetone and 12.0 g of purified water and mix them in a beaker withan overhead stirrer. Weigh 40 g of the Eudragit and pour into thesolvent mixture slowly in portions to prevent lump formation. Stir untila clear solution is made. Then weigh 6 g of triethyl citrate and addinto the clear solution and keep stirring until a homogeneous solutionis made. Mix around 60 g of placebo tablets with about 80 of the 750 mgimmediate release tablets and coat with the coating mixture.

Example 26: Sustained Release Tablets

The blend of the formulation is prepared in the same manner as theimmediate release tablets (e.g. sieving, blending, and compression).Other preparations are acceptable, such as wet granulation, fluidizedbed, high shear granulation, etc. The formulation includes drugmodifying release excipients. These excipients include but not limitedto HPMC (hydroxy propyl methylcellulose or hypromellose), methacrylicpolymers, polyvinyl acetate, and povidone. The amount of drug releasemodifying excipient ranges from about 10% to about 80% in theformulation. The drug release profile ranges from 0 to 4 hours, 0 to 6hours, 0 to 8 hours, 0 to 12 hours, 0 to 24 hours, 2 to 4 hours, 2 to 6hours, etc. In some embodiments, the formulations are coated with theOpadry coatings after direct compression. Example sustained releaseformulations are listed below.

TABLE 18 Sustained Release Tablet Amount per tablet (mg) %, w/w Compound1 or Compound 2 250.0 33.3 Mannitol 90.0 12.0 Prosolv HD 90 177.5 23.7Sodium Stearyl Fumarate 7.5 1.0 Methocel K4M 225.0 30.0 Total 750 100

TABLE 19 Sustained Release Tablet Amount per tablet (mg) %, w/w Compound1 or Compound 2 250.0 33.3 Mannitol 112.5 15.0 Prosolv HD 90 230.0 30.7Sodium Stearyl Fumarate 7.5 1.0 Methocel K 100 LV 150.0 20.0 Total 750.0100

Example 27: Ointment Compositions

A non-limiting example of an ointment composition is presented in Table20.

TABLE 20 PEG Ointment Compositions Formula # Components (% w/w) AA BB CCCompound 1 or Compound 2 5 5 5 Stearyl Alcohol 4 — 1 PEG 3350 40 40 36Butylated hydroxyl Toluene 0.1 0.1 0.1 (BHT) Dimethicone (Q7-9120Silicone — — 1 Fluid, 350 CST) Brij 721 — — 3 PEG 400 50.9 54.9 53.9

Example 28: Gel Compositions

A non-limiting example of a gel composition is presented in Table 21.

TABLE 21 Wt % Components 1 2 3 Compound 1 or 1 1.5 1 Compound 2Transcutol P — 10 — Propylene glycol 10 — 10 PEG 400 40 40 15 Benzylalcohol 1 1.0 2 Gelling agent 1-2 1-1 1-2 Glycerin 10 10 10 EDTAdisodium 0.5 0.5 0.5 Methylparaben 0.17 0.17 0.17 Propylparaben 0.030.03 0.03 pH adjuster pH adjusted to 7 pH adjusted to 7 pH adjusted to 7Purified water q.s.a.d. q.s.a.d. q.s.a.d. (or buffer)

Example 29: Cream Compositions

A non-limiting example of a cream composition is presented in Table 22.

TABLE 22 Formula # Components A B C D Transcutol P 25 25 25 25 Propyleneglycol 20 20 20 20 Mineral Oil 5 5 5 5 Dimethicone 5 5 5 5 (Q7-9120Silicon Fluid 100 CST) BHT 0.1 0.1 0.1 0.1 Brij 72 1.0 — 1.2 — Brij 7211.8 — 1.8 — Glycerul Monosterate SE — 10 — — Sorbitan Monosterate — — —2.5 Pamulen TR1 — 0.3 — — Stearyl Alcohol 1.0 — 5 5 Cetyl Alcohol 0.5 —3 3 EDTA disodium 0.05 0.05 0.05 0.05 Methylparaben 0.17 0.17 0.17 0.17Propylparaben 0.03 0.03 0.03 0.03 Carbopol Ultrez 10 0.4 0.2 — — 4% NaOHqs pH 7 qs pH 7 — — Purified Water qsad qsad — — 20 mM Phosphate — —qsad qsad Buffer (pH 7) Compound 1 or 3% (w/w) 3% (w/w) 3% (w/w) 3%(w/w) Compound 2

Example 30: Identification of Metabolic Pathways

Compound 1 metabolites formed during incubation of Compound 1 with: rat,dog, monkey, and human liver microsomes; rat, dog, and humanhepatocytes; as well as those generated in vivo and isolated from ratbile and rat and dog plasma were investigated.

Materials

Male Sprague-Dawley rats, male beagle dogs, and mixed pool humancryopreserved hepatocytes were purchased from Celsis (Woburn, Mass.).Fresh human hepatocyctes were purchased from Life Technologies (lot00583558, male; Carlsbad, Calif.). KB media was from Celsis. UDPGA,β-NADPH and tryptophan blue was from Sigma Chemical.

Compound 2 (2 mg/kg) was administered intravenously (IV) to fasted ratsas a solution in 0.9% saline via a bolus injection into the jugular vein(2 mg/mL; 2 mL/kg).

Microsomes

To determine the qualitative metabolic profile, 30 μM of Compound 1 wasincubated aerobically with rat, dog, monkey, or human liver microsomes(1 mg/mL). The incubations were performed in phosphate buffer at pH 7.4,37° C., with the reaction initiated by the addition of P-NADPH, GSHand/or UDPGA (1, 5 and 2 mM final concentration, respectively). Thereaction was terminated by the addition of 3 times incubation volume ofacetonitrile after 60 minutes. The sample was centrifuged and thesupernatant was transferred, nitrogen blow dried and reconstituted in50% acetonitrile in water for LC/MS analysis.

Hepatocytes

Rat, dog, monkey, or human hepatocytes were thawed according to thesupplier's instructions. Cells were counted using the Trypan Bluemethod, and then diluted to 1×10⁶ viable cells/ml with KB medium.Compound 1 was tested at 30 μM and incubated for up to 2 hours in rathepatocytes and 4 hours in dog, monkey or human hepatocytes at 37° C.Reactions were terminated with addition of 3 times incubation volume ofacetonitrile, centrifuged, and supernatants were transferred, nitrogenblow dried and reconstituted in 50% acetonitrile in water for LC/MSanalysis.

Rat Bile Duct Cannulation

Rats with surgically placed bile duct and jugular vein cannula werepurchased from Charles River Laboratories and allowed to acclimate for 2days. Compound 2 was intravenously dosed (2 mg/kg) to three rats as asolution in 0.9% saline (2 mg/mL; 1 mL/kg). Bile samples were collectedat time-points 0-2, 2-5 and 5-8, and urine samples were collected attime-points 0-4 and 4-8 hrs post-dose in 8 mL scintillation vials andstored at −40° C. until analysis.

Plasma Extraction of Compound 1

Concentrations of Compound 1 in rat plasma were determined by LC-MS/MSafter protein precipitation. Rat plasma (100 μL) was treated with 400 μLof internal standard solution (ISTD, buspirone in acetonitrilecontaining 1.5% acetic acid) to precipitate proteins. Samples werevortexed and then centrifuged for 10 min at approximately 4,000 rpm(3700×g) at 4° C. (Beckman centrifuge, Brea, Calif.).

Instruments

LC/MS/MS analysis was carried out with a Sciex API-4000Qtrap tandem massspectrometer (Applied Biosystems/Life Technologies, Carlsbad, Calif.)interfaced to a high-performance liquid chromatography system consistingof an Agilent 1200 series pump (Foster City, Calif.) and a LEAPTechnologies PAL autoinjector (Carrboro, N.C.).

LC-MS/MS Procedure

Parent/metabolite analysis was conducted in the positive ion mode (ESI)by multiple reaction monitoring of parent. The mobile phases contained10 mM ammonium acetate in water with 0.05% formic acid (solvent A) and10 mM ammonium acetate in 50% acetonitrile/50% methanol with 0.05%formic acid (solvent B). For Compound 1, the flow rate was maintained at1 mL/min and the total run time was 2.5 min. Analytes were separated ona YMC ODS-AQ column (2.1×150 mm; 3 μm) and eluted with a linear gradientas follows:

-   -   1. mobile phase was held for 0.5 min at 5% B,    -   2. B was increased from 5% to 95% over the next 0.2 min,    -   3. B was held constant for 1.3 min at 95%, and    -   4. B was returned to the initial gradient conditions.

GSH adducts were investigated in microsomes, hepatocytes and rat bileusing several methods including the neutral loss of pyroglutamate (129Da) as well as a precursor ion scans of Compound 1 (105 Da, 318 Da and438 Da).

For metabolite quantitation, the flow rate was maintained at 0.25 mL/minand the total run time was 60 min. Analytes were separated using alinear gradient as follows:

-   -   1. mobile phase was held for 5 min at 5% B,    -   2. B was increased from 5% to 95% over the next 45 min.    -   3. B was held constant for 5 min at 95%, and    -   4. B was returned to the initial gradient conditions

Results

The following metabolites were observed:

TABLE 23 Metabolites of Compound 1 Metabolite Structure MetaboliteDescription M1

Glucuronidation of Compound 1 M2

Glucuronidation of Compound 1 plus oxidation M3

Oxidation of phenyl ring of benzyl group. M4

Oxidation of phenyl ring of benzyl group. M5

Oxidation of biphenyl

Glucuronides, including M1, are (direct and indirect) the majormetabolites observed in liver microsomes, hepatocytes and rat bile.Mono-oxidation metabolites on the phenyl ring (M3 and M4) and thebiphenyl ring are also observed in rat, dog and human liver microsomes,hepatocytes as well as in rat bile. An oxidated glucuronide (M2) wasobserved in liver microsomes (rat, dog & human), hepatocytes (dog) andbile (rat). Neither the primary amine (i.e. decarboxylation) nor itssecondary metabolites (glycosylation or glucuronidation) are observed inplasma (rat & dog), rat bile or urine. No GSH adduct was observed in thein vitro screening assay, in rat, dog or human hepatocytes, and humanmicrosomes. In vivo, there is neither GSH adduct is observed in ratbile, nor any metabolites in rat urine. In vivo, metabolites M1 and M4AUC values are at less than 10% of the parent, Compound 1.

Example 31. Pharmacokinetics in Sprague-Dawley Rats

The pharmacokinetics of Compound 1 and Compound 2 was assessed in maleand female Sprague-Dawley rats (225-300 g at 10 weeks).

Compound 2 (1 mg/kg) was administered intravenously (IV) to fasted rats(n=3) as a solution in purified water via a bolus injection into thejugular vein (1 mg/mL; 1 mL/kg). Compound 1 (10 mg/kg) was administeredorally (PO), to fasted (for at least 12 hours) or non-fasted rats (n=2or 3) as a solution in 0.5% methylcellulose unless otherwise noted viaan oral gavage to the stomach (3.33 mg/mL; 3 mL/kg). Non-fasted animalswere fasted for at least 12 hours and then were given food ad libitumfor 1 hour prior to dosing. Fasted animals were dosed 1, 10, 30, 100 or300 mg/kg Compound 1 as a solution in 0.5% methylcellulose via an oralgavage to the stomach (0.69 mg/mL, 10 mg/mL, 33.3 mg/mL or 100 mg/mL)(n=2 or 3 per dose group). In a gastrointestinal study, rats wereanesthetized and Compound 1 was administered directly to the duodenum,jcjunum or ilcum at 1 mg/kg in 0.5% methylcellulose.

Capsules were also administered with Compound 2. Capsules were size 9gelatin (Torpac, San Diego, Calif.).

TABLE 24 Capsule and Compound 2 weight. Capsule # Capsule Weight (mg)Compound 2 Weight (mg) 1 9.86 3.21 2 10.53 3.05 3 10.2 3.18 4 9.38 3.06

Blood samples (approximately 300 μL total blood) were taken from eachrat via the jugular vein cannula at pre-dose, and an initial time of 5or 15 min and then various time points up to 24 hours post-dose (10-11samples per animal). Samples were collected on wet ice in tubescontaining potassium EDTA. After each blood draw, the cannula wasflushed with an equivalent volume of heparinized saline (0.1 mL at 40units/mL). Plasma samples, prepared by centrifugation of whole blood,were stored frozen (−80° C.) prior to analysis.

All other reagents were of analytical grade.

After intravenous (IV) administration (1 mg/kg), the clearance (5.1 to5.2 μg·hr/mL) was low for both male and female rats: the volume ofdistribution was moderate and was approximately two-fold total bodywater. The half-life was 3.8 and 3.2 hours in males and females,respectively. No significant gender related PK differences were notedafter intravenous dosing. In addition, Compound 1 suspension inmethylcellose was dosed as an oral gavage (PO) to fasted male/femaleanimals at doses of 1, 10, 30, 100, and 300 mg/kg. Subsequent to a 1mg/kg dose, the resulting maximal plasma concentrations were 0.4 and 0.3μg/mL (Cmax) in male and female rats, respectively. The mean doseadjusted exposure (AUC₀₋₂₄/D) was 2.1 and 1.9 g·hr/mL in males andfemales respectively, which resulted in an apparent bioavailability of63% (male) and 58% (female) for Compound 1. As noted above (IV dosing at1 mg/kg), no significant gender differences were observed after a POadministration. Upon oral administration at 10 mg/kg to fasted animals,the dose adjusted AUC value_((AUC0-24/D)) was 2.4 and 2.7 μg·hr/mL inmale and female rats, respectively. These data suggest that between POdoses of 1 and 10 mg/kg, Compound 1 exposure is dose proportional whenadministered as a suspension. However, as the dose was increased to 30,100 and 300 mg/kg, the resulting exposure increased in a greater thandose proportional manner. Upon oral administration at 1 mg/kg to fedanimals, the exposure (AUC₀₋₂₄) was 1.6 and 1.4 μg·hr/mL in male andfemale rats, respectively. The trend of slightly lower exposure in fedanimals vs. fasted was also observed at 10 mg/kg. Two additional studieswere completed to assess the oral pharmacokinetics of Compound 2formulated in capsule form, or as a free acid in suspension. The dosenormalized exposure of the free acid, sodium salt and sodium salt in agelatin capsule (10 mg/kg) was 5.1, 2.4 and 2.1 μg·hr/mL, respectively.Regional absorption of Compound 1 shows good oral bioavailability alongthe entire gastrointestinal tract.

Example 32. Pharmacokinetics in Male Beagle Dogs

The pharmacokinetics of Compound 1 and Compound 2 was assessed in maleBeagle dogs.

Dosing of male Beagle dogs (n=3) was performed. Compound 1 or Compound 2was dosed intravenously at 2 mg/kg (fasted) and orally (fasted ornon-fasted) at 5 mg/kg. In fasted animals, food was withheld a minimumof 12 hours prior to dosing and then returned 4 hours post-dose.Non-fasted animals were allowed to feed 1 hour prior to dosing adlibitum. Compound 2 capsules were given by oral gavage in the fastedstate. Plasma samples were collected by Perry Scientific studypersonnel.

The capsules are noted in Table 25 below. Capsules were size 0 gelatin(Capsugel, Peapack, N.J.). Tablet formulations are noted in Example 24.

TABLE 25 Capsule and Compound 2 weight. Capsule # Capsule Weight (mg)Compound 2 Weight (mg) 1 90.64 55.24 2 88.41 55.31 3 91.55 64.91 4 87.5760.26

For the clinical drug product oral solution of active agent (1.67mg/mL), sucralose (0.5% w/w) and cherry flavor (0.5% w/w) is added tothe formulation as a sweetener and taste masking agent, respectively toa 0.5% methocel aqueous solution.

Blood samples (approximately 1 mL total blood) were taken from each dogat pre-dose, and an initial time of 5 or 15 min and then various timepoints up to 24 hours post-dose (10-11 samples per animal). Samples werecollected on wet ice in tubes containing potassium EDTA. After eachblood draw, the cannula was flushed with an equivalent volume ofheparinized saline (0.1 mL at 40 units/mL). Plasma samples, prepared bycentrifugation of whole blood, were stored frozen (−80° C.) prior toanalysis.

All other reagents were of analytical grade.

After intravenous administration of 2 mg/kg Compound 2, the compoundshowed a systemic clearance value of 10.5 mL/min/kg, an estimated volumeof distribution value of 0.6 L/kg, and a 3.9 hr terminal half-life. Oraladministration of 5 mg/kg Compound 2 in the fasted state showed anapparent oral bioavailability of 63%, with a C_(max) value of 5.5 μg/mL,while the fed state showed an apparent oral bioavailability of 60%. WhenCompound 2 was dosed as a capsule form, the AUC decreased 2.5-fold, andhad an apparent oral bioavailability of 24%. When Compound 1 was dosed,it had reduced exposure, with an apparent oral bioavailability of 19%.Two different tablet formulations and a human dose solution were alsoinvestigated, and each showed similar AUC values and an apparent oralbioavailability of 100, 52 and 57%, respectively. The pharmacokineticresults suggest that Compound 2 has systemic exposure and food haslittle effect on the absorption in dog.

Example 33. Establishment of a CHO Cell Line Stably Expressing HumanLPA₁

A 1.1 kb cDNA encoding the human LPA₁ receptor was cloned from humanlung. Human lung RNA (Clontech Laboratories, Inc. USA) was reversetranscribed using the RETROscript kit (Ambion, Inc.) and the full-lengthcDNA for human LPA₁ was obtained by PCR of the reverse transcriptionreaction. The nucleotide sequence of the cloned human LPA₁ wasdetermined by sequencing and confirmed to be identical to the publishedhuman LPA₁ sequence (An et al. Biochem. Biophys. Res. Commun. 231:619(1997). The cDNA was cloned into the pCDNA5/FRT expression plasmid andtransfected in CHO cells using lipofectamine 2000 (Invitrogen Corp.,USA). Clones stably expressing human LPA₁ were selected using hygromycinand identified as cells that show Ca-influx in response to LPA.

Example 34. Generation of Cells Transiently Expressing Human LPA₂

A vector containing the human LPA₂ receptor cDNA was obtained from theMissouri S&T cDNA Resource Center (www.cdna.org). The full-length cDNAfragment for human LPA₂ was obtained by PCR from the vector. Thenucleotide sequence of the cloned human LPA₂ was determined bysequencing and confirmed to be identical to the published human LPA₂sequence (NCBI accession number NM_004720). The cDNA was cloned into thepCDNA3.1 expression plasmid and transfected into B103 cells (InvitrogenCorp., USA) by seeding cells in a 96-well poly-D-lysine coated plate at30,000-35,000 cells per well together with 0.2 μl lipofectamine 2000 and0.2 μg of the LPA₂ expression vector. Cells were cultured overnight incomplete media before being assayed for LPA-induced Ca-influx.

Example 35. Establishment of a CHO Cell Line Stably Expressing HumanLPA₃

A vector containing the human LPA₃ receptor cDNA was obtained from theMissouri S&T cDNA Resource Center (www.cdna.org). The full-length cDNAfragment for human LPA₃ was obtained by PCR from the vector. Thenucleotide sequence of the cloned human LPA₃ was determined bysequencing and confirmed to be identical to the published human LPA₃sequence (NCBI accession number NM_012152). The cDNA was cloned into thepCDNA5/FRT expression plasmid and transfected in CHO cells usinglipofectamine 2000 (Invitrogen Corp., USA). Clones stably expressinghuman LPA₃ were selected using hygromycin and identified as cells thatshow Ca-influx in response to LPA.

Example 36. LPA1 and LPA3 Calcium Flux Assays

Human LPA₁ or LPA₃ expressing CHO cells are seeded at 20,000-45,000cells per well in a 96-well poly-D-lysine coated plate one or two daysbefore the assay. Prior to the assay, the cells are washed once with PBSand then cultured in serum-free media overnight. On the day of theassay, a calcium indicator dye (Calcium 4, Molecular Devices) in assaybuffer (HBSS with Ca²⁺ and Mg²⁺ and containing 20 mM Hepes and 0.3%fatty-acid free human serum albumin) is added to each well andincubation continued for 1 hour at 37° C. 10 μl of test compound in 2.5%DMSO are added to the cells and incubation continued at room temperaturefor 30 minutes. Cells are the stimulated by the addition of 10 nM LPAand intracellular Ca²⁺ measured using the Flexstation 3 (MolecularDevices). IC₅₀s are determined using Graphpad prism analysis of drugtitration curves.

Example 37. LPA2 Calcium Flux Assay

BT-20 human breast cancer cells are seeded at 25,000-35,000 cells perwell in 150 μl complete media on Poly-D-Lysine coated black-wallclear-bottom plates. Following an overnight culture, cells are washedonce with PBS then serum starved for 4-6 hours prior to the assay. Onthe day of the assay, a calcium indicator dye (Calcium 5, MolecularDevices) in assay buffer (HBSS with Ca²⁺ and Mg²⁺ and containing 20 mMHepes and 0.3% fatty-acid free human serum albumin) is added to eachwell and incubation continued for 15 minutes at 37° C. 25 μl of testcompounds in 2.5% DMSO are added to the cells and incubation continuedat 37° C. for 15 minutes. Cells are the stimulated by the addition of100 nM LPA and intracellular Ca²⁺ measured using the Flexstation 3(Molecular Devices). IC₅₀s are determined using Symyx Assay Exploreranalysis of drug titration curves.

Illustrative in vitro biological data is presented in the Table below.

TABLE 26 Calcium Flux IC₅₀ Data Ca Flux IC₅₀ (μM) LPA₁ LPA₂ LPA₃ LPA₄LPA₅ Compound 1 A C C C C S-enantiomer of Compound 1 A ND C ND NDRacemic Compound 1 A ND C ND ND A = less than 0.2 uM, B = 0.2-1.0 uM,and C = greater than 1 uM; ND = assay not performed

Example 38. LPA1 Chemotaxis Assay

Chemotaxis of the A2058 human melanoma cells was measured using theNeuroprobe ChemoTx® System plates (8 μm pore size, 5.7 mm diametersites). The filter sites were coated with 0.001% fibronectin (Sigma) in20 mM Hepes, pH 7.4 and allowed to dry. A2058 cells were serum-starvedfor 24 hours, then harvested with Cell Stripper and resuspended in DMEMcontaining 0.1% fatty-acid-free bovine serum albumin (BSA) to aconcentration of 1×10⁶/ml. Cells were mixed with an equal volume of testcompound (2×) in DMEM containing 0.1% fatty-acid-free BSA and incubatedat 37° C. for 15 minutes. LPA (100 nM in DMEM containing 0.1%fatty-acid-free BSA) or vehicle was added to each well of the lowerchamber and 50 μl of the cell suspension/test compound mix was appliedto the upper portion of the ChemoTx plate. Plates were incubated at 37°C. for three hours and then the cells removed from the upper portion byrinsing with PBS and scraping. The filter was dried then stained withHEMA 3 Staining System (Fisher Scientific). The absorbance of the filterwas read at 590 nM and IC₅₀s were determined using Symyx Assay Explorer.

Compound 1, inhibited LPA-driven chemotaxis (IC₅₀ less than 100 nM) ofhuman A2058 melanoma cells

Example 39: Bleomycin-Induced Lung Fibrosis Model in Mice

Female C57Bl/6 mice (Harlan, 25-30 g) are housed 4 per cage, given freeaccess to food and water and allowed to acclimate for at least 7 daysprior to test initiation. After the habituation phase, mice are lightlyanesthetized with isoflurane (5% in 100% O₂) and administered withbleomycin sulfate (0.01-5 U/kg, Henry Schein) via intratrachealinstillation (Cuzzocrea S et al. Am J Physiol Lung Cell Mol Physiol.2007 May; 292(5):L 1095-104. Epub 2007 Jan. 12.). Mice are returned totheir cages and monitored daily for the duration of the experiment. Testcompound or vehicle is delivered po, ip or sc daily. The route andfrequency of dosing is based on previously determined pharmacokineticproperties. All animals are sacrificed using inhaled isoflurane 3, 7,14, 21 or 28 days after bleomycin instillation. Following sacrifice,mice are intubated with a 20 gauge angiocatheter attached to a 1 mlsyringe. Lungs are lavaged with saline to obtain bronchoalveolar lavagefluid (BALF) and then removed and fixed in 10% neutral buffered formalinfor subsequent histopathological analysis. BALF is centrifuged for 10min at 800×g to pellet the cells and the cell supernatant removed andfrozen at −80° C. for subsequent protein analysis using the DC proteinassay kit (Biorad, Hercules, Calif.) and soluble collagen analysis usingSircol (Biocolor Ltd, UK). BALF is analyzed for concentrations ofinflammatory, pro-fibrotic and tissue injury biomarkers includingtransforming growth factor β1, hyaluronic acid, tissue inhibitor ofmetalloproteinase-1, matrix matelloproteinase-7, connective tissuegrowth factor and lactate dehydrogenase activity, using commerciallyavailable ELISA. The cell pellet is re-suspended in PBS. Total cellcounts are then obtained using a Hemavet hematology system (DrewScientific, Wayne, Pa.) and differential cells counts are determinedusing Shandon cytospin (Thermo Scientific, Waltham, Mass.). Lung tissueis stained using hematoxylin and eosin (H&E) and trichrome and lungfibrosis. is determined by semiquantitative histopathological scoring(Ashcroft T. et al. J. Clin. Path. 1988; 41; 4, 467-470) using lightmicroscopy (10× magnification) and quantitative, computer-assisteddensitometry of collagen in lung tissue sections using light microscopy.The data are plotted using Graphpad prism and statistical differencesbetween groups determined.

In the acute setting (3 day), Compound 1 significantly reduced totalprotein and collagen concentrations in broncheoalveolar lavage fluid(BALF). In a 7-day bleomycin model Compound 1 reduced BALF collagen,protein, TGFβ1, MMP-7, hyaluronan, and inflammatory cell influx. In thechronic setting (14 day bleomycin model), Compound 1 decreased totallung collagen when dosed either propylactically (day 0-day 14) ortherapeutically (day 3-day 14).

Example 40: Mouse Carbon Tetrachloride (CCl₄)-Induced Liver FibrosisModel

Female C57BL/6 mice (Harlan, 20-25 g) housed 4/cage are given freeaccess to food and water and allowed to acclimate for at least 7 daysprior to test initiation. After the habituation phase, mice receive CCl₄(1.0 ml/kg body weight) diluted in corn oil vehicle (100 μL volume) viai.p. injection twice a week for 8 weeks. (Higazi, A. A. et al., Clin ExpImmunol. 2008 April; 152(1):163-73. Epub 2008 Feb. 14.). Control micereceive an equivalent volume of corn oil vehicle only. Test compound orvehicle is delivered po, ip or sc daily. At the end of the study (8weeks after first i.p. injection of CCl₄), mice are sacrificed usinginhaled isoflurane and blood is drawn via cardiac puncture forsubsequent analysis of ALT/AST levels. The liver is harvested, and onehalf of the liver is frozen at −80° C. and the other half is fixed in10% neutral buffered formalin for histological assessment of liverfibrosis using light microscopy (10× magnification). Liver tissuehomogenates are analyzed for collagen levels using Sircol (Biocolor Ltd,UK). Fixed Liver tissue is stained using hematoxylin and eosin (H&E) andtrichrome and liver fibrosis is determined by quantitative,computer-assisted densitometry of collagen in liver tissue sectionsusing light microscopy. Plasma and liver tissue lysates are alsoanalyzed for concentrations of inflammatory, pro-fibrotic and tissueinjury biomarkers including transforming growth factor β1, hyaluronicacid, tissue inhibitor of metalloproteinase-1, matrixmatelloproteinase-7, connective tissue growth factor and lactatedehydrogenase activity, using commercially available ELISA. Theresulting data are plotted using Graphpad prism and statisticaldifferences between groups determined.

In this experiment, Compound 1 significantly reduced liver weightincrease and collagen deposition in the liver as compared to theuntreated group.

Example 41: Mouse Intravenous LPA-Induced Histamine Release

A mouse intravenous LPA-induced histamine release model is utilized todetermine the in vive potency of LPA₁ and LPA₃ receptor antagonists.Female CD-1 mice (weighing 25-35 grams) are administered compound (i.p.,s.c. or p.o.) in a volume of 10 ml/kg 30 minutes to 24 hours prior tointravenous LPA challenge (300 μg/mouse in 0.1% FAF BSA). Immediatelyfollowing LPA challenge mice are placed into an enclosed Plexiglaschamber and exposed to an isoflurane for a period of 2 minutes. They areremoved, decapitated and trunk blood collected into tubes containingEDTA. Blood is then centrifuged at 10,000×g for 10 minutes at 4° C.Histamine concentrations in the plasma are determined by EIA. Drugconcentrations in plasma are determined by mass spectrometry. The doseto achieve 50% inhibition of blood histamine release is calculated bynonlinear regression (Graphpad Prism) and plotted as the ED₅₀. Theplasma concentration associated with this dose is plotted as the EC₅₀.

Example 42: Mouse Dermal Vascular Leak Assay

Female BALB/c mice (Harlan) weighing 20-25 grams were given free accessto standard mouse chow and water and were allowed to acclimate for twoweeks prior to study initiation. Compound 1 was prepared in watervehicle at a concentration of 3 mg/ml and delivered by oral gavage at avolume of 10 ml/kg to yield a dose of 30 mg/kg. Three hours followingdose, mice were placed into a restraining device and given Evan's bluedye intravenously by tail vein injection (0.2 ml of a 0.5% solution).Mice were then anesthetized using 3% isoflurane anaesthesia to allow forintradermal injection of LPA (30 μg in 20 μl 0.1% fatty acid free BSA).Thirty minutes after LPA injection mice were sacrificed by CO₂inhalation and the skin removed from the challenge site and placed into2 ml formamide for overnight extraction of Evan's blue dye.

Following extraction, a 150 μl aliquot of formamide for each tissuesample was placed into a 96 well plate and read at 610 nm using aphotospectometer. The resulting data (OD units) were plotted usingGraphPad Prizm. In this experiment Compound 1 reduced LPA-induced Evan'sblue dye leak into the skin.

Example 43: Mouse Model of Bleomycin-Induced Scleroderma

A mouse model of bleomycin-induced scleroderma was used to evaluate theeffect of Compound 1 in skin fibrosis. Methods were adapted from(Yamamoto, T et al. The Journal of Investigative Dermatology, 112:456-462, 1999). Female C57Bl/6 mice were anesthetized with isoflurane(3.0-3.5% in 100% O₂) and two areas shaved bilaterally on the lowerdorsolateral region. BLM (1-10 μg in 100 μl) prepared in sterilefiltered PBS was administered subcutaneously to each shaved region oncedaily for 5 to 7 days per week for a total of 4 weeks (28 days).

Compound 1 was prepared in water vehicle and delivered orally twice aday on weekdays and once daily on weekends.

On day 28 all animals were sacrificed. The dorsolateral skin removed,trimmed of adherent subcutaneous fat and an 8 mm biopsy punch was usedto collect two skin samples from each, subject. One sample submerged in10% neutral buffered formalin and submitted for histological analysis.The second sample was frozen at −80° C. for further processing ofcollagen content using either Sircol or hydroxyproline methods.

FIGS. 14 and 15: Results of assay on mouse model of bleomycin-inducedscleroderma using Compound 1. FIG. 14 shows dermal thickness. FIG. 15shows collagen content. #P<0.05 versus PBS; *P<0.05 versus BLM; ANOVA.Compound 1 reduced both the dermal thickness and collagen content.

Example 44: Mouse Unilateral Ureteral Obstruction Kidney Fibrosis Model

Female C57BL/6 mice (Harlan, 20-25 g) housed 4/cage will be given freeaccess to food and water and allowed to acclimate for at least 7 daysprior to test initiation. After the habituation phase, mice undergounilateral ureteral obstruction (UUO) surgery or sham to left kidney.Briefly, a longitudinal, upper left incision is performed to expose theleft kidney. The renal artery is located and 6/0 silk thread is passedbetween the artery and the ureter. The thread is looped around theureter and knotted 3 times insuring full ligation of ureter. The kidneyis returned to abdomen, the abdominal muscle is sutured and the skin isstapled closed. Mice are returned to their cages and monitored daily forthe duration of the experiment. Test compound or vehicle is deliveredpo, ip or sc daily. The route and frequency of dosing is based onpreviously determined pharmacokinetic properties. All animals aresacrificed using inhaled isoflurane 4, 8 or 14 days after UUO surgery.Following sacrifice blood is drawn via cardiac puncture, the kidneys areharvested and one half of the kidney is frozen at −80° C. and the otherhalf is fixed in 10% neutral buffered formalin for histologicalassessment of kidney fibrosis using light microscopy (10×magnification). Kidney tissue homogenates are analyzed for collagenlevels using Sircol (Biocolor Ltd, UK). Fixed kidney tissue is alsostained using hematoxylin and eosin (H&E) and trichrome and kidneyfibrosis is determined by quantitative, computer-assisted densitometryof collagen in liver tissue sections using light microscopy and collagencontent in kidney lysate. Plasma and kidney tissue lysates are alsoanalyzed for concentrations of inflammatory, pro-fibrotic and tissueinjury biomarkers including transforming growth factor β1, hyaluronicacid, tissue inhibitor of metalloproteinase-1, and plasminogen activatorinhibitor-1, using commercially available ELISA. The resulting data areplotted using Graphpad prism and statistical differences between groupsdetermined.

In this experiment, Compound 1 reduced total kidney collogen, collagenType 1, transforming growth factor β1, hyaluronic acid, tissue inhibitorof metalloproteinase-1 and plasminogen activator inhibitor-1 compared tountreated group.

Example 45: Clinical Trial in Humans with Idiopathic Pulmonary Fibrosis(IPF) Purpose

The purposes of this study is to assess the efficacy of treatment withCompound 1, or a pharmaceutically acceptable salt thereof (e.g. Compound2), compared with placebo in patients with idiopathic pulmonary fibrosis(IPF) and to assess the safety of treatment with Compound 1, or apharmaceutically acceptable salt thereof (e.g. Compound 2), comparedwith placebo in patients with IPF.

The primary outcome variable is the absolute change in percent predictedforced vital capacity (FVC) from baseline to Week 72.

Secondary outcome measures include: composite outcomes of importantIPF-related events; progression-free survival; categorical assessment ofabsolute change in percent predicted FVC from baseline to Week 72;change in Shortness-of-Breath from baseline to Week 72; change inpercent predicted hemoglobin (Hb)-corrected carbon monoxide diffusingcapacity (DLco) of the lungs from baseline to Week 72; change in oxygensaturation during the 6 minute walk test (6MWT) from baseline to Week72; change in high-resolution computed tomography (HRCT) assessment frombaseline to Week 72; change in distance walked in the 6MWT from baselineto Week 72.

Criteria

Patients eligible for this study include those patients that satisfy thefollowing inclusion criteria: diagnosis of IPF; 40 to 80 years of age;FVC≧50% predicted value; DLco≧35% predicted value; either FVC orDLco≦90% predicted value; no improvement in past year; able to walk 150meters in 6 minutes and maintain saturation≧83% while on no more than 6L/min supplemental oxygen.

Patients are excluded from this study if they satisfy any of thefollowing criteria: unable to undergo pulmonary function testing;evidence of significant obstructive lung disease or airwayhyper-responsiveness; in the clinical opinion of the investigator, thepatient is expected to need and be eligible for a lung transplant within72 weeks of randomization; active infection; liver disease; cancer orother medical condition likely to result in death within 2 years;diabetes; pregnancy or lactation; substance abuse; personal or familyhistory of long QT syndrome; other IPF treatment; unable to take studymedication; withdrawal from other IPF trials.

Patients are orally dosed with either placebo or an amount of Compound1, or a pharmaceutically acceptable salt thereof (e.g. Compound 2), (1mg/day-1000 mg/day). The primary outcome variable will be the absolutechange in percent predicted FVC from Baseline to Week 72. Patients willreceive blinded study treatment from the time of randomization until thelast patient randomized has been treated for 72 weeks. A Data MonitoringCommittee (DMC) will periodically review safety and efficacy data toensure patient safety.

After week 72, patients who meet the Progression of Disease (POD)definition, which is a ≧10% absolute decrease in percent predicted FVCor a ≧15%/absolute decrease in percent predicted DLco, will be eligibleto receive permitted IPF therapies in addition to their blinded studydrug. Permitted IPF therapies include corticosteroids, azathioprine,cyclophosphamide and N-acetyl-cysteine.

The examples and embodiments described herein are illustrative andvarious modifications or changes suggested to persons skilled in the artare to be included within this disclosure. As will be appreciated bythose skilled in the art, the specific components listed in the aboveexamples may be replaced with other functionally equivalent components,e.g., diluents, binders, lubricants, fillers, and the like.

1.-18. (canceled)
 19. A process for the preparation of1-{4′-[3-methyl-4-((R)-1-phenyl-ethoxycarbonylamino)-isoxazol-5-yl]-biphenyl-4-yl}-cyclopropanecarboxylicacid (Compound 1) comprising Step (1) and Step (2): (1) treatment of acompound of Formula XVIII with diphenylphosphoryl azide in the presenceof (R)-(+)-1-phenylethanol:

Wherein, R¹ is C₁-C₆ alkyl; to provide a compound of Formula X:

Or (1) reacting a compound of Formula VII:

Wherein, x is a leaving group; With a compound of Formula VIII:

Wherein, R¹ is C₁-C₆ alkyl; and B is a boronic acid or boronate ester;in the presence of a coupling catalyst, a suitable base, and in asuitable solvent, to provide a compound of Formula X:

or (1) reacting a compound of Formula IX:

wherein, B is a boronic acid or boronate ester; With a compound ofFormula XII:

wherein, R¹ is C₁-C₆ alkyl; and X is a leaving group; In the presence ofa coupling catalyst, a suitable base, and in a suitable solvent, toprovide a compound of formula X:

and (2) hydrolysis of the ester moiety of the compound of Formula X toprovide Compound
 1. 20. The process according to claim 19, wherein step(2) comprises treatment of the compound of Formula X with sodiumhydroxide in a suitable solvent followed by a pH adjustment.